Industrial Products – Philippe Gabant, SYNGULON SA

Abstract for “Controlled Growth of Microorganisms”

It can be used to regulate the growth microbial cells. Certain embodiments provide genetically engineered cells for microbial growth that can produce bacteriocins. Some embodiments allow microbial cells to be contained in a desired environment. In some embodiments, the contaminating microbial cell types are neutralized. One embodiment allows a first type of microbial cells to regulate the growth of another type of microbial cells in order to maintain the desired ratio.

Background for “Controlled Growth of Microorganisms”

Since the dawn of time, humans have used microbial organisms for product generation, such as in the production of cheese, beer and wine. Over the centuries, microbial organisms-mediated processes were studied and scaled up, often by controlling fermentation conditions, or identifying phenotypic traits of microbial bacterias.

Many products are currently made using microbial organisms. Microbial organisms can be grown in controlled, sterile environments in laboratories and pharmaceutical manufacturing processes. However, feedstocks that are used in various industrial processes that involve microorganisms may not be sterile and could contain many strains or species. Genetically engineered microorganisms are not suitable for industrial processes such as those that involve feedstocks, or are exposed to microorganisms in their environment, which could potentially contaminate the culture, and may also include changing environmental conditions. These microorganisms have been designed to manage their own growth, that of other microorganisms, and/or respond to environmental changes. These microorganisms can be grown in non-sterile and less rigidly controlled feedstocks. These microorganisms are useful in the production of consistent, robust products across a variety of environments and feedstocks.

“One embodiment of this invention includes a first cell that contains a nucleic acids encoding a secreted Bacteriocin. This nucleic acids controls the growth of another microbial cells. A second cell is also provided with a nucleic Acid which confers resistance. The first cell has been genetically engineered so that the activity or expression of the nucleic a which confers resistance can be controlled. This embodiment states that the activity or expression of the nucleic acids which confer resistance to the bacteriocin can be reduced to a point where the first microbial cells are neutralized by the bacteriocin. Some aspects of this embodiment state that the first microbial cells have been genetically engineered in order to produce a desired product. Some aspects of this embodiment state that the secreted Bacteriocin has been chosen to maintain at least one environment in a culture where the desired product is being produced by the first microbial cells. Some aspects of this embodiment state that the culture contains at least one invading microorganism. Some aspects of this embodiment state that at least one condition of the cultivation is controlling the growth of the second microorganism. The second microbial organism may be a common contaminate in the culture. Some aspects of this embodiment state that the second microbial cells are different species, genuses, or strains from the first. Some aspects of this embodiment state that the microbial cells also contain a nucleic acids encoding a second secreted Bacteriocin. This nucleic is responsible for controlling the growth of a third cell. Also, it confers resistance against the second bacteriocin. The first microbial cells have been genetically engineered to regulate the activity or expression of the nucleic. The bacteriocin kills a second microbial cells according to certain aspects of this embodiment. Some aspects of this embodiment state that the bacteriocin decreases the growth rate for the second microbial cells. Some aspects of this embodiment state that the bacteriocin stops the growth of the second microorganism. A regulatable promoter controls transcription of the nucleic acids conferring resistance to the Bacteriocin, according to certain aspects of this embodiment. Some aspects of this embodiment state that the activity of the polypeptide encoded in the nucleic acids conferring resistance to the Bacteriocin can be controlled. Some aspects of this embodiment state that the nucleic acids encoding the Bacteriocin are on the chromosomes of the microbial cells. Some aspects of this embodiment state that the nucleic acids conferring resistance to the Bacteriocin are on a plasmid. Some aspects of this embodiment state that the nucleic acids encoding the Bacteriocin are on the chromosomes of microbial cells, while the nucleic acids conferring resistance to the Bacteriocin are on plasmids. Some aspects of this embodiment state that the nucleic acids encoding the Bacteriocin and those conferring resistance to the Bacteriocin are located on one or more different plasmids. Some aspects of this embodiment state that the first microbial cells are selected from among bacteria, yeast, or algae.

“Another embodiment described herein comprises a method for controlling the growth a second microbe in a culture medium. This method involves the cultivation of a first microbial cells in conditions where the first microbial cells produce bacteriocin sufficient to control growth of the second microbe. Some aspects of this embodiment require that the culture be maintained continuously for at least 30 consecutive days. This could include at least 30, 35-40, 45, 55, 60 and 65, 70. 75, 85, 95, 90. 95, 100. 110, 130, 140. 150, 160. 170. 180. 190. 200. 250. 350. 400. 450. The method also includes the detection of at least one change within the culture medium. This change may include the presence or an increase in activity of a third cell. Reengineering the first cell to produce a second form of bacteriocin in response to this change is necessary to control growth of the third cell.

“Another embodiment is disclosed that allows for the detection of a presence, absence or amount of a chemical in a culture. This can be done by culturing a first genetically-engineered microbial cells that contain a bacteriocin and a genetically regulateable promoter. The regulatable promor controls transcription so that (a) transcription is driven by the regulatable promotor in the presence of a molecule but not in its absence; (b) transcription is driven in the absence but not in its presence. This can include determining the amount of genomic nucleic acids in the first microbial cells from the culture. This can include determining the presence, absence, and quantity of a particular nucleic acids sequence characteristic of the first microbe. The method can also be used to compare the amount of the nucleic acids sequences characteristic of the first microbial cells to the quantity of a reference sequence.

“Another embodiment described herein comprises a genetically engineered virus that contains a nucleic acids conferring resistance against bacteriocin. In this case, the activity or expression of the nucleic acids is modified to respond to the presence, concentration, or absence of a component in a feedstock. The vector may also include a nucleic acids that encode the bacteriocin, according to certain aspects. The vector may also contain a nucleic acids that encode a product, according to certain aspects of this embodiment.

“Another embodiment described herein is a kit that can include a plurality strains of genetically engineered microorganisms. Each strain has been genetically modified to allow expression or activity of a nucleic acids which confer resistance to a different type of bacteriocin to being regulated.

“Another embodiment described herein involves a method for identifying at minimum one bacteriocin that modulates the growth in at least 1 microbial cells in an industrial culture media. This includes contacting the medium with a composition or medium containing the at most one bacteriocin and determining if the at-least one bacteriocin has any desired effect on the growth in the at-least one microbial cells. The method, according to certain aspects of this embodiment, involves contacting the industrial medium with at least one bacteria produced by a first microbial cells as described herein. Some aspects of this embodiment state that the at least one bacteriocin created by the first microorganism is contained in the supernatant from the culture containing the first microbial cells. The method also includes the creation of a genetically engineered microorganism to produce at most one bacteriocin that has been shown to have a desired effect upon the growth of at least one of the microbial cells. Some aspects of this embodiment state that the least one microbial cells is an organism that is a common invader in the industrial culture medium. Some aspects of this embodiment state that the least one microbial cells is an organism that has invaded an industrial culture.

“Another embodiment described herein involves a system for neutralizing unwanted microbial organisms within a culture medium. The system may include a first environment that includes a culture medium and a second environment that contains a second microorganism that secretes two or three different bacteriocins. In this second environment, immunity modulators are provided for each of these bacteriocins. The second environment is physically isolated from the first environmental so that the second organism cannot move between the environments. The system can also include a first microorganism in the culture medium. This means that the first microbial species does not secrete any of the two or three different bacteriocins and the first microbial entity is not neutralized by any one of the two or three different bacteriocins. This embodiment states that the first microbial organism is not genetically modified. Some aspects of this embodiment state that the first microbial organism ferments an ingredient of the culture medium. Some aspects of this embodiment state that the culture medium is decontaminated by the first microbial organism. Some aspects of this embodiment state that the first microbial organism performs photosynthesis. The substrate used in the photosynthesis is also included. Some aspects of this embodiment state that the second environment is separated by the first environment using at least one of a membrane or mesh or filter. However, it is not permeable for the second microbial species. Some aspects of this embodiment state that the second microbial organism secretes at most three bacteriocins. These include at least 3, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, and 20 bacteriocins. Some aspects of this embodiment state that the second environment contains at least one third microbial organism. This organism also secretes some bacteriocins. Some aspects of this embodiment state that the third microbial organism secretes at most 2 bacteriocins. For example, at least 2, 3, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 19 or 20 bacteriocins. A method for storing a feedstock is another embodiment described herein. This could include providing a feedstock and providing a first microorganism that secretes two or three different bacteriocins. Then, the method involves contacting the feedstock with these bacteriocins and then storing it for the desired time. According to certain aspects of this embodiment contact the feedstock using the bacteriocins means that the feedstock is contacted with the microbial organism. Some aspects of this embodiment state that contacting the feedstock using the bacteriocins involves putting the microbial species in fluid communication with it, while keeping the feedstock and microbial organisms physically separated. This ensures that the bacteriocins can contact the feedstock but not the microbial. Some aspects of this embodiment state that the separation is maintained by at minimum one or more membranes, meshes, filters, or valves that are permeable to two or more different types of bacteriocins but not the first microbial species. The method may also include fermenting the feedstock using a second microbial organism before or concurrently with contact with the bacteriocins. Some aspects of this embodiment state that the fermentation can be used to produce a desired component or remove an undesirable component from the feedstock. Some aspects of this embodiment state that the desired time period is at least one month. This could be one, two or three, four, five to six months, seven to eight weeks, nine to nine, ten, eleven, twelve, or twelve. Some aspects of this embodiment state that the desired time period is at least six months. This could be six, seven or eight, nine, ten, eleven, twelve, or twelve. Some aspects of this embodiment state that the first microbial organism must secrete at least three bacteriocins. These could include at least 3, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

“Genetically engineered microbial organisms can be found in some of the examples. Some embodiments of the microbial organisms can be engineered to limit the growth of the microbial populations in environments such as those that use a feedstock. As used herein, ?neutralizing? Activity (and variations on the same root word), can refer to either arresting microbial reproduction or cytotoxicity. It is possible to engineer microbial organisms to produce bacteriocins. These secreted polypeptides can neutralize microorganisms. Certain bacteriocins can be repelled by microbial organisms that have bacteriocin immunity modators. In some embodiments, the first microbial organism is designed to secrete bacteriocins. Some embodiments select the bacteriocins based on the type and location of the microbial cells, the composition of the medium or the geographic location. This is to target specific contaminating organisms that are associated with the medium or geographical location. Other microbial species that exhibit desired characteristics can produce bacteriocin immune modulators and thus survive in the presence bacteriocins. Unwanted microbial species (for example, contaminants, organisms that have lost a desired characteristic, or organisms involved in an industrial process, but whose growth or production is not desirable under the prevailing conditions), fail to produce bacteriocin immuno modulators and are therefore neutralized by the Bacteriocins.

“Microbial Organisms”

“Genetically engineered microorganisms can be provided according to certain aspects. Genetically engineered microbial organism is used herein. ?microorganism,? These root terms can be used in various variations (such as pluralizations) and include genetic modification of any naturally occurring species, fully synthetic prokaryotic and eukaryotic unicellular species as well as Archae species. This expression can be used to refer to cells of bacteria, fungal, and algae.

Examples of microorganisms that could be used according to the embodiments are, but not limited to, yeast, bacteria, and algae. Full synthetic microorganism genomes are also possible to be synthesized and transferred into single microbial cell, to create synthetic microorganisms that can self-replicate continuously (see Gibson et. al. (2010), “Creation of Bacterial Cells Controlled by Chemically Synthesized Genes”, Science 329: 52?56. This article is hereby included by reference in its entirety. In some cases, the microorganism can be fully synthesized. The desired combination of genetic elements can be assembled onto a chassis to create a fully or partially synthetic microorganism. Wright et al. also provide a description of genetically engineered microorganisms for industrial purposes. (2013). Building-in biosafety to synthetic biology. Microbiology 159: 1221-1235.”

“A variety of bacterial strains and species can be used in accordance to embodiments herein. Genetically modified variants or synthetic bacteria based upon a?chassis?” An example of a species is provided. Exemplary bacteria that can be used to make industrially useful characteristics according to the embodiments are, among others, Bacillus species (for instance Bacillus subtilis and Bacillus), Streptomyces species and Streptomyces species.

“A variety can be used to make yeast strains and species, including genetically modified varieties and synthetic yeast that is based on a?chassis. An example of a species is provided. Exemplary yeast can be provided with industrially-applicable characteristics.

“A variety can be used to create synthetic algae using genetically modified strains or species based on a ‘chassis. It is possible to create a new species. Some embodiments of the algae include photosynthetic microalgae. Examples of algae species that could be useful in biofuels and can be used according to embodiments herein include Botryococcus braunii and Chlorella species. Many algae species can also be used for fertilizer products, food production, waste neutralization, environmental remediation, carbohydrate manufacturing (for instance, biofuels).

“Bacteriocins”

“As used herein, ?bacteriocin,? This root term and variants of it, refers to a protein that is secreted in host cells and can neutralize at most one other cell than the host cell in which the polypeptide was made. As used herein, ?bacteriocin? Also includes a cell-free version or chemically synthesized form of this polypeptide. A cell that expresses one particular immune modulator. A cell that expresses a specific?immunity modulator? is immune to the neutralizing effect of a particular bacteriocin (or group of bacteriocins). The bacteriocins are able to neutralize the effects of bacteriocins on a cell that produces it and/or other microbial organisms, provided they don’t produce an immunity modulator. A host cell can secrete bacteriocins to inhibit growth or cytotoxicity in order to affect a variety of microbial organisms. The translational machinery (e.g. A ribosome, etc. A microbial cell. A bacteriocin can be chemically synthesized in some embodiments. A polypeptide precursor can be used to make some bacteriocins. To obtain the bacteriocin polypeptide, the precursor polypeptide can be cleaved (for example by a protease). In some embodiments, the precursor polypeptide is used to make a bacteriocin. A bacteriocin may be a polypeptide which has been subject to post-translational modifications such as cleavage or addition of one or more functional group.

“?Antibiotic,? “?Antibiotic” and other variations of this root term refer to a metabolite or intermediate in a metabolic pathway that can kill or arrest growth of at least one microbe. Microbial cells can produce some antibiotics, such as bacteria. Some antibiotics can also be made chemically. It is clear that antibiotics and bacteriocins can be synthesized chemically.

“Neutralizing activity can be described as the arrest of microbial reproduction or cytotoxicity. Some bacteriocins can be cytotoxic (e.g. ?bacteriocide? ?bacteriocide? Some bacteriocins may inhibit the reproduction of microbial species (e.g. ?bacteriostatic? ?bacteriostatic?

“It should be noted that there have been non-bacteriocin strategies to target different microbial organisms. KAMORAN is one example. A chemical has been suggested to target Lactic Acid Bacteria family bacteria (LAB) (see Union Nationale des Groupements de Distillateurs D’Alcool (2005)?Kamoran). It should be noted that phage was also proposed to target LAB-family bacteria (see U.S. Pub. No. 2010/0330041). It should be noted that pesticides are being considered to target a variety of contaminating microorganisms (see McBride and al.,?Contamination management in low cost open algae ponds for biofuels production?). Industrial Biotechnology 10: 221-227 (2014). However, bacteriocins have many advantages over pesticides and phages. Bacteriocins are able to prevent potentially toxic runoff from a feedstock. Bacteriocins may have a higher effectiveness against certain undesired microorganisms than chemicals, pesticides or phages. Logarithmic growth can produce bacteriocins, which can easily be scaled up or down. This is in contrast to phages and chemical/pesticide system, which can be less scalable. Bacteriocins, for example, can be used to control which organisms are neutralized. This is useful for avoiding the neutralization of industrially valuable microbial organisms in culture media. Phage production at industrial scale can be challenging. They can also be very difficult to control. However, bacteriocins according to some embodiments can be part of an industrial process. They can contain gene and/or control a fermenting process via bacteriostatic activities. Immunity control can also be used to adjust the susceptibility of microorganisms involved in an industrial process. Bacteriocins are typically low in toxicity for industrial uses such as food for animals or humans. It is possible that bacteriocins according to some embodiments herein could be used as a food additive.

“In some embodiments, a specific neutralizing activity (e.g. “Some embodiments have a particular neutralizing activity (e.g., arrest of microbial replication) that is determined by the type of microbial regulation desired. In some cases, microbial cells can be engineered to express a particular combination or bacteriocins. In some embodiments, for example, microbial cells can be engineered to express specific bacteriocins depending on how they are regulated. For example, if the contaminating cells must be killed, at least one cytotoxic-bacteriocin may be provided in some embodiments. One or more bacteriocins or combinations thereof that are effective against contaminants found in particular cultures, geographic locations, or particular types of culture grown in particular geographical areas may be used in some embodiments. A bacteriocin which inhibits microbial reproduction may be used in some embodiments. Many bacteriocins have the ability to neutralize microbial organisms, regardless of their ecological niche. In some cases, it is desirable to have a specific spectrum of bacteriocin activities. To this end, a bacteriocin may be selected from a host that is similar or identical to the microbial organism/s being targeted by the bacteriocin.

“In some embodiments, one, or more, bacteriocin activities may be selected before culture growth and one or two microbial organisms are engineered for the desired culture environment. Some embodiments allow for bacteriocins to be chosen based on their ability neutralize invading organisms that are likely to try to grow in the culture. Another embodiment is where strain A produces intermediate A and strain B converts it into intermediate B. In this case, the equilibrium can shift to favor strain B through the generation of a profile of bacteriocin activities that favors strain A. A lack of intermediateA will cause the equilibrium profile to favor strain A. This profile will inhibit or prevent growth of strain B. One or more bacteriocin activities can be selected depending on the conditions of an existing culture. If certain invaders are found in a culture environment, the?neutralizer’ is selected. It is possible to engineer microorganisms to produce bacteriocins in order to neutralize identified invaders. Some embodiments add genetically engineered cells capable of producing bacteriocins to an existing culture to re-equilibrate it, such as when a certain microbial type grows in the microbial culture. Some embodiments add genetically engineered cells that make bacteriocins to an existing culture to neutralize all, or substantially all, of the microbial cells within the culture. This is done to, for example, to remove an industrial culture from the culture environment to allow for the introduction of a new industrial culture.

“For example, some embodiments may have anti-fungal activity (such anti-yeast activity). There are many bacteriocins that have anti-fungal activity. For example, bacteriocins from Bacillus have been shown to have neutralizing activity against yeast strains (see Adetunji and Olaoye (2013) Malaysian Journal of Microbiology 9: 130-13, hereby incorporated by reference in its entirety), an Enterococcus faecalis peptide (WLPPAGLLGRCGRWFRPWLLWLQ SGAQY KWLGNLFGLGPK, SEQ ID NO: 1) has been shown to have neutralizing activity against Candida species (see Shekh and Roy (2012) BMC Microbiology 12: 132, hereby incorporated by reference in its entirety), and bacteriocins from Pseudomonas have been shown to have neutralizing activity against fungi such as Curvularia lunata, Fusarium species, Helminthosporium species, and Biopolaris species (Shalani and Srivastava (2008) The Internet Journal of Microbiology. Volume 5, Number 2. DOI: 10.5580/27dd?accessible on the worldwide web at archive.ispub.com/journal/the-internet-journal-of-microbiology/volume-5-number-2/screening-for-antifungal-activity-of-pseudomonas-fluorescens-against-phytopathogenic-fungi.html#sthash.d0Ys03UO.1DKuT1US.dpuf, hereby incorporated by reference in its entirety). Botrycidin AJ1316 is an example (see Zuber, P. et al. (1993) Peptide Antibiotics. In Bacillus subtilis and other Gram-Positive Bacteria : Biochemistry, Physiology and Molecular Genetics ed Sonenshein et., pp. 897-916, American Society for Microbiology. hereby incorporated in its entirety). and Alirin B1 (see Shenin, et al. (1995) Antibiot Khimioter 51: 3-7. Antifungal activities have been demonstrated in B. subtilis. In some cases, such as those that require neutralization of a fungal organism, the bacteriocin may contain at least one botrycidin A1316 or alirin C1.

“For example, some embodiments make it desirable to have bacteriocin activity within cyanobacteria cultures. Some embodiments provide bacteriocins to neutralize cyanobacteria. Some embodiments provide bacteriocins to kill invading microorganisms that are commonly found in cyanobacteria cultures. A wide range of cyanobacteria species have been found to contain clusters of conserved polypeptides of bacteriocin. Wang et. al. reported that at least 145 clusters of putative bacteriocin genes have been found in at least 43 species of cyanobacteria. Genome Mining demonstrates the widespread occurrence of gene clusters coding bacteriocins within Cyanobacteria. PLoS ONE 6(7) : e22384, hereby incorporated in its entirety by reference. Table 1.2 shows exemplary cyanobacteria Bacteriocins. These are the SEQ ID NO’s 422, 422, 426, 428 and 30.

“In some embodiments, the host cells themselves are a microbial cell. Bacteriocins can neutralize cells from a different strain or species than the host cell in some embodiments. If the cells are not equipped with an immune modulator, some embodiments allow bacteriocins to neutralize cells of the same strain or species as the host cells. The skilled artisan will be able to tell that bacteriocins are capable of neutralizing both host and non-host microorganisms. Bacteriocins are capable of neutralizing cells other than those in which they were produced. This means that poison molecules can only be killed the specific cell in which they were produced.

“A variety of bacteriocins were identified and characterized. Exemplary bacteriocins may be classified as “class I” without being restricted by any particular theory. bacteriocins that are subject to post-translational modifications. bacteriocins which are usually unmodified. Furthermore, the exemplary bacteriocins from each class can be classified into different subgroups. Table 1.1 is adapted by Cotter, P. D., and others. Are bacteriocins a viable alternative to antibiotics. Nature Reviews Microbiology 11 (95-105) is hereby included by reference in its entirety.

“Bacteriocins are not limited by any one theory. They can neutralize a target microbial cells in many ways.” A bacteriocin, for example, can penetrate a cell wall to depolarize it and interfere with respiration.

“TABLE 1.1\nClassification of Exemplary Bacteriocins\nGroup Distinctive feature Examples\nClass I (typically modified)\nMccC7- Is covalently attached MccC7-C51\nC51-type to a carboxy-\nbacteriocins terminal aspartic acid\nLasso peptides Have a lasso structure MccJ25\nLinear azole- or Possess heterocycles MccB17\nazoline-containing but not other\npeptides modifications\nLantibiotics Possess lanthionine Nisin,\nbridges planosporicin,\nmersacidin,\nactagardine,\nmutacin 1140\nLinaridins Have a linear Cypemycin\nstructure and contain\ndehydrated amino acids\nProteusins Contain multiple Polytheonamide\nhydroxylations, A\nepimerizations and\nmethylations\nSactibiotics Contain sulphur-?- Subtilosin A,\ncarbon linkages thuricin CD\nPatellamide-like Possess heterocycles Patellamide A\ncyanobactins and undergo\nmacrocyclization\nAnacyclamide- Cyclic peptides consisting Anacyclamide\nlike of proteinogenic amino A10\ncyanobactins acids with prenyl\nattachments\nThiopeptides Contain a central pyridine, Thiostrepton,\ndihydropyridine or nocathiacin\npiperidine ring as I, GE2270 A,\nwell as heterocycles philipimycin\nBottromycins Contain macrocyclic a Bottromycin A2\nmidine, a decarboxylated\ncarboxy-terminal\nthiazole and carbon-\nmethylated amino\nacids\nGlycocins Contain S-linked Sublancin 168\nglycopeptides\nClass II (typically unmodified or cyclic)\nIIa peptides Possess a conserved Pediocin PA-1,\n(pediocin YGNGV motif enterocin\nPA-1-like (in which N represents CRL35,\nbacteriocins) any amino acid) carnobacteriocin\nBM1\nIIb peptides Two unmodified ABP118,\npeptides are required lactacin F\nfor activity\nIIc peptides Cyclic peptides Enterocin AS-48\nIId peptides Unmodified, linear, MccV, MccS,\nnon-pediocin-like, epidermicin NI01,\nsingle-peptide bacteriocins lactococcin A\nIIe peptides Contain a serine-rich MccE492, MccM\ncarboxy-terminal\nregion with a non-ribosomal\nsiderophore-type modification”

“Numerous bacteriocins may be used according to the embodiments. Table 1.2 shows examples of bacteriocins. In certain embodiments, at most one bacteriocin is comprised of a sequence of polypeptides from Table 1.2. Some bacteriocins are able to function as pairs of molecules, as shown in Table 12.2. It will be clear that, unless stated otherwise, functional bacteriocins are essentially a pair of molecules. If a functional bacteriocin is used, or if it provides a bacteriocin? The like is discussed herein. Functional bacteriocin pair are included alongside bacteriocins which function separately. Refer to Table 1.2 for information on?organisms that are of origin? The alternative names and/or strain information of organisms that produce the indicated bacteriocin are listed in parentheses.

“Embodiments” herein include peptides as well as proteins that are identical to the bacteriocins listed in Table 1.2. “Identity” is a broad term that can be used to describe any combination of nucleic acid or protein sequence homology, or three-dimensional homology. The term “identity” can refer to nucleic acids, protein sequence homology, or three-dimensional homology. There are many methods that can be used to determine the nucleic acid sequence homology or three-dimensional homology of polypeptides. These techniques are used to determine the degree of identity between a sequence, domain or model and a target sequence or domain. There are many functional bacteriocins that can include features of the bacteriocins described herein. This allows for a wide range of identity to the bacteriocins listed in Table 1.2. A bacteriocin may have at least 50% identity in some embodiments. For example, 51%/52%/53%/54%/56%/57%/58%/69%/70%/74%/76%/77%/78%/78%/79%/78%/79%/78%/79%/79%/80%, 80%, 85%, 85%, 86%/87%, 89%, 89%, 99%, 91% 92% 93% 94% 95% 96% 97% 98%, 98%, 99.9% identity to any of Table 1.2. BLAST software (Altschul S. F. et al.) can be used to determine percent identity. (1990)?Basic Local Alignment Search Tool. J. Mol. Biol. 215:403-410, accessible on the world wide web at blast.ncbi.nlm.nih.gov) with the default parameters.”

“In some embodiments, the polynucleotide that encodes a bacteriocin according to this invention is provided. The polynucleotide may be contained within an expression vector in some embodiments. In some embodiments, the expression vector or polynucleotide is found in a microbial cells. Table 1.2 shows examples of polynucleotide sequences that encode the polypeptides. Table 1.2 shows examples of polynucleotides that are based on reverse translation. SEQ ID NOS: 341 to 419 (odd numbers SEQ ID) are examples. A skilled artisan will quickly understand that a polypeptide can be encoded by more than one polynucleotide. The genetic code can be degenerate and codon usage may vary depending on the organism where the gene product is expressed. A polynucleotide that encodes a bacteriocin may be selected according to the codon usage of the organism that possesses the gene product. A polynucleotide that encodes bacteriocin may be codon optimized according to the specific organism that carries it.

“While Table 1.2 contains bacteriocins that are naturally occurring, the skilled artisan will recognize that there are variants of these bacteriocins, naturally-occurring Bacteriocins, and variants thereof. Synthetic bacteriocins may also be used in some of the embodiments. These variants may have a higher or lower level of cytotoxic activity or growth inhibition activity than the wild type protein in some embodiments. Many motifs have been identified as characteristics of bacteriocins. The N-terminal consensus sequence of class IIa Bacteriocins is represented by the motif YGXGV (SEQID NO: 2), in which X can be any amino acid residue. In some embodiments, the N-terminal sequence of a synthetic bacteriocin is at least 50% identical to SEQID NO: 2. A synthetic bacteriocin may include a N-terminal sequence that includes SEQ ID NO. 2. Some class IIb bacteriocins also contain a GxxxG motif. Without being limited by any particular theory, it is believed that the GxxxG motif can mediate association between helical proteins in the cell membrane, for example to facilitate bacterioncin-mediated neutralization through cell membrane interactions. In some embodiments, the motif in bacteriocin facilitates interaction with the cell membrane. The bacteriocin may contain a GxxxG motif in some embodiments. A helical structure can be added to a bacteriocin containing a GxxxG-motive. This structure is not the only one that is described in this article. Structures that have substantially the exact same effect on microorganisms as any of the bacteriocins provided herein are also included in the definition of?bacteriocin.

“As used herein ?bacteriocin polynucleotide? Refers to a polynucleotide that encodes a bacteriocin. In some embodiments, at least one bacteriocin is present in the host cell.

“Bacteriocin immunity modulators”

“Table 2 shows examples of bacteriocin-based immunity modulators. The immunity modulators shown in Table 2 are natural-occurring. However, a skilled artisan will recognize that there are other naturally-occurring immunity modators than those listed in Table 2. Synthetic immunity modulators or variants can also be used.

“In certain embodiments, an immunity modulator, or combination thereof, confers immunity against a specific bacteriocin or class or category of Bacteriocins or a particular combination of Bacteriocins. Table 2 lists examples of bacteriocins for which immunity modulators may confer immunity. Table 2 lists the ‘organism of origin? These immunity modulators are exemplary examples. However, they can be easily expressed in other naturally occurring, genetically modified or synthetic microorganisms to produce the desired bacteriocin activity, in accordance with certain embodiments. As such, the term “immunity modulator” is used herein. This term refers to not only structures described herein but also to structures that have substantially similar effects to the?immunity modator? Structures described herein include fully synthetic immunity modulators and immunity modulators which provide immunity to bacteriocins functionally equivalent to those disclosed herein.

Table 2 shows examples of polynucleotide sequences that encode the polypeptides. Table 2 lists the examples of polynucleotide sequences that encode the polypeptides. A skilled artisan will quickly understand that the genetic code can be degenerate. A polynucleotide that encodes a bacteriocin immune modulator may be selected according to the codon usage of an organism expressing this gene. A polynucleotide that encodes a bacteriocin immune modulator may be codon optimized according to the specific organism that possesses the modulator. There are many functional immunity modulators that can include features from the immunity modulators described herein. This allows for a wide range of combinations, which provides a high degree of identity to the immunity modators listed in Table 2. An immunity modulator may have at least 50% identity in some embodiments. For example, 51%-52%, 53%-54%, 56%-57%, 58%-59%, 69%-69%, 70%, 71%-72%, 74%-73%, 74%-76%, 77% and 78%, 77%-77%, 78%-79%, 78%-79%, 78%-78%, 79%-79%, 80% or 90% identity to any of the polypeptides listed in Table 2.

“TABLE 2\nExemplary bacteriocin immunity modulators\nPoly- Poly-\npeptide nucleotide\nSEQ Polypeptide Organism SEQ\nID NO: Name Sequence of origin ID NO: Polynucleotide Sequence\n452 Microcin MSYKKLY Escherichia 453 ATGAGTTATAAAAAAC\nH47 QLTAIFSLP coli TGTACCAATTGACGGCT\nimmunity LTILLVSLS ATATTTAGTTTACCTCT\nmodulator SLRIVGEG TACTATCTTATTGGTTT\nMchI NSYVDVFL CACTTTCATCCCTTCGG\nSFIIFLGFIE ATTGTTGGCGAAGGGA\nLIHGIRKIL ATTCTTATGTTGACGTT\nVWSGWKN TTTCTAAGCTTTATAAT\nGS ATTTCTTGGTTTTATTG\nAGCTGATTCATGGGATT\nCGAAAGATTTTGGTCTG\nGTCAGGCTGGAAAAAC\nGGAAGTTAA\n454 Colicin-E3 MGLKLDLT Escherichia 455 ATGGGACTTAAATTGG\nimmunity WFDKSTED coli ATTTAACTTGGTTTGAT\nmodulator FKGEEYSK AAAAGTACAGAAGATT\n(Colicin-E3 DFGDDGSV TTAAGGGTGAGGAGTA\nchain B) MESLGVPF TTCAAAAGATTTTGGAG\n(ImmE3) KDNVNNG ATGACGGTTCAGTTATG\n(Microcin- CFDVIAEW GAAAGTCTAGGTGTGC\nE3 VPLLQPYF CTTTTAAGGATAATGTT\nimmunity NHQIDISD AATAACGGTTGCTTTGA\nmodulator) NEYFVSFD TGTTATAGCTGAATGG\nYRDGDW GTACCTTTGCTACAACC\nATACTTTAATCATCAAA\nTTGATATTTCCGATAAT\nGAGTATTTTGTTTCGTT\nTGATTATCGTGATGGTG\nATTGGTGA\n456 Colicin-E1 MSLRYYIK Escherichia 457 ATGAGCTTAAGATACTA\nimmunity NILFGLYC coli CATAAAAAATATTTTAT\nmodulator TLIYIYLIT TTGGCCTGTACTGCACA\n(ImmE1) KNSEGYYF CTTATATATATATACCT\n(Microcin- LVSDKML TATAACAAAAAACAGC\nE1 YAIVISTIL GAAGGGTATTATTTCCT\nimmunity CPYSKYAI TGTGTCAGATAAGATG\nmodulator) EYIAFNFIK CTATATGCAATAGTGAT\nKDFFERRK AAGCACTATTCTATGTC\nNLNNAPVA CATATTCAAAATATGCT\nKLNLFMLY ATTGAATACATAGCTTT\nNLLCLVLA TAACTTCATAAAGAAA\nIPFGLLGLF GATTTTTTCGAAAGAAG\nISIKNN AAAAAACCTAAATAAC\nGCCCCCGTAGCAAAATT\nAAACCTATTTATGCTAT\nATAATCTACTTTGTTTG\nGTCCTAGCAATCCCATT\nTGGATTGCTAGGACTTT\nTTATATCAATAAAGAAT\nAATTAA\n458 Cloacin MGLKLHIH Escherichia 459 ATGGGGCTTAAATTAC\nimmunity WFDKKTEE coli ATATTCATTGGTTTGAT\nmodulator FKGGEYSK AAGAAAACCGAAGAGT\nDFGDDGSV TTAAAGGCGGTGAATA\nIESLGMPL CTCAAAAGACTTCGGT\nKDNINNG GATGATGGTTCTGTCAT\nWFDVEKP TGAAAGTCTGGGGATG\nWVSILQPH CCTTTAAAGGATAATAT\nFKNVIDISK TAATAATGGTTGGTTTG\nFDYFVSFV ATGTTGAAAAACCATG\nYRDGNW GGTTTCGATATTACAGC\nCACACTTTAAAAATGTA\nATCGATATTAGTAAATT\nTGATTACTTTGTATCCT\nTTGTTTACCGGGATGGT\nAACTGGTAA\n460 Colicin-E2 MELKHSIS Escherichia 461 ATGGAACTGAAACATA\nimmunity DYTEAEFL coli GTATTAGTGATTATACC\nmodulator EFVKKICR GAGGCTGAATTTCTGG\n(ImmE2) AEGATEED AGTTTGTAAAAAAAAT\n(Microcin- DNKLVREF ATGTAGAGCTGAAGGT\nE2 ERLTEHPD GCTACTGAAGAGGATG\nimmunity GSDLIYYP ACAATAAATTAGTGAG\nmodulator) RDDREDSP AGAGTTTGAGCGATTA\nEGIVKEIKE ACTGAGCACCCAGATG\nWRAANGK GTTCAGATCTGATTTAT\nSGFKQG TATCCTCGCGATGACAG\nGGAAGATAGTCCTGAA\nGGGATTGTCAAGGAAA\nTTAAAGAATGGCGAGC\nTGCTAACGGTAAGTCA\nGGATTTAAACAGGGCT\nGA\n462 Colicin-A MMNEHSID Citrobacter 463 ATGATGAATGAACACT\nimmunity TDNRKAN freundii CAATAGATACGGACAA\nmodulator NALYLFIII CAGAAAGGCCAATAAC\n(Microcin- GLIPLLCIF GCATTGTATTTATTTAT\nA immunity VVYYKTPD AATAATCGGATTAATAC\nmodulator) ALLLRKIA CATTATTGTGCATTTTT\nTSTENLPSI GTTGTTTACTACAAAAC\nTSSYNPLM GCCAGACGCTTTACTTT\nTKVMDIYC TACGTAAAATTGCTACA\nKTAPFLALI AGCACTGAGAATCTCCC\nLYILTFKIR GTCAATAACATCCTCCT\nKLINNTDR ACAACCCATTAATGACA\nNTVLRSCL AAGGTTATGGATATTTA\nLSPLVYAA TTGTAAAACAGCGCCTT\nIVYLFCFR TCCTTGCCTTAATACTA\nNFELTTAG TACATCCTAACCTTTAA\nRPVRLMAT AATCAGAAAATTAATC\nNDATLLLF AACAACACCGACAGGA\nYIGLYSIIFF ACACTGTACTTAGATCT\nTTYITLFTP TGTTTATTAAGTCCATT\nVTAFKLLK GGTCTATGCAGCAATTG\nKRQ TTTATCTATTCTGCTTC\nCGAAATTTTGAGTTAAC\nAACAGCCGGAAGGCCT\nGTCAGATTAATGGCCA\nCCAATGACGCAACACT\nATTGTTATTTTATATTG\nGTCTGTACTCAATAATT\nTTCTTTACAACCTATAT\nCACGCTATTCACACCAG\nTCACTGCATTTAAATTA\nTTAAAAAAAAGGCAGT\nAA\n464 Colicin-Ia MNRKYYF Escherichia 465 ATGAACAGAAAATATT\nimmunity NNMWWG coli ATTTTAATAATATGTGG\nmodulator WVTGGYM TGGGGATGGGTGACGG\nLYMSWDY GGGGATATATGCTGTA\nEFKYRLLF TATGTCATGGGATTATG\nWCISLCGM AGTTTAAATACAGATTA\nVLYPVAK CTGTTCTGGTGTATTTC\nWYIEDTAL TCTCTGCGGAATGGTTT\nKFTRPDFW TGTATCCGGTTGCAAAA\nNSGFFADT TGGTATATTGAAGATAC\nPGKMGLLA AGCTCTAAAATTTACCC\nVYTGTVFI GGCCTGATTTCTGGAAC\nLSLPLSMIY AGCGGTTTTTTTGCTGA\nILSVIIKRLS TACACCTGGAAAAATG\nVR GGGTTGCTTGCGGTTTA\nTACGGGTACTGTTTTCA\nTATTATCTCTTCCGTTA\nAGTATGATATATATTCT\nTTCTGTTATTATAAAAA\nGGCTGTCTGTAAGATAG\n466 Colicin-Ib MKLDISVK Escherichia 467 ATGAAACTGGATATATC\nimmunity YLLKSLIPI coli TGTAAAGTATTTACTGA\nmodulator LIILTVFYL AAAGCCTGATACCAAT\nGWKDNQE CCTCATTATTCTTACAG\nNARMFYAF TTTTTTATCTGGGATGG\nIGCIISAITF AAAGATAACCAGGAAA\nPFSMRIIQK ATGCAAGAATGTTTTAT\nMVIRFTGK GCGTTCATCGGATGCAT\nEFWQKDFF TATCAGTGCCATTACTT\nTNPVGGSL TTCCTTTTTCAATGAGG\nTAIFELFCF ATAATACAGAAAATGG\nVISVPVVAI TAATAAGGTTTACAGG\nYLIFILCKA GAAAGAATTCTGGCAA\nLSGK AAAGACTTCTTTACAAA\nTCCAGTTGGCGGAAGC\nTTAACTGCAATATTTGA\nATTATTCTGTTTCGTTA\nTATCAGTTCCTGTGGTT\nGCCATTTACTTAATTTT\nTATACTCTGCAAAGCCC\nTTTCAGGAAAATGA\n468 Colicin-N MHNTLLEK Escherichia 469 ATGCACAATACACTCCT\nimmunity IIAYLSLPG coli CGAAAAAATCATCGCA\nmodulator FHSLNNPP TACCTATCCCTACCAGG\n(Microcin- LSEAFNLY ATTTCATTCATTAAACA\nN immunity VHTAPLAA ACCCGCCCCTAAGCGA\nmodulator) TSLFIFTHK AGCATTCAATCTCTATG\nELELKPKS TTCATACAGCCCCTTTA\nSPLRALKIL GCTGCAACCAGCTTATT\nTPFTILYIS CATATTCACACACAAAG\nMIYCFLLT AATTAGAGTTAAAACC\nDTELTLSS AAAGTCGTCACCTCTGC\nKTFVLIVK GGGCACTAAAGATATT\nKRSVFVFF AACTCCTTTCACTATTC\nLYNTIYWD TTTATATATCCATGATA\nIYIHIFVLL TACTGTTTCTTGCTAAC\nVPYRNI TGACACAGAACTAACC\nTTGTCATCAAAAACATT\nTGTATTAATAGTCAAAA\nAACGATCTGTTTTTGTC\nTTTTTTCTATATAACAC\nTATATATTGGGATATAT\nATATTCACATATTTGTA\nCTTTTGGTTCCTTATAG\nGAACATATAA\n470 Colicin-E8 MELKNSIS Escherichia 471 ATGGAACTGAAAAACA\nimmunity DYTETEFK coli GCATTAGTGATTACACT\nmodulator KIIEDIINCE GAAACTGAATTCAAAA\n(ImmE8) GDEKKQD AAATTATTGAAGACATC\n(Microcin- DNLEHFIS ATCAATTGTGAAGGTG\nE8 VTEHPSGS ATGAAAAAAAACAGGA\nimmunity DLIYYPEG TGATAACCTCGAGCATT\nmodulator) NNDGSPEA TTATAAGTGTTACTGAG\nVIKEIKEW CATCCTAGTGGTTCTGA\nRAANGKSG TCTGATTTATTACCCAG\nFKQG AAGGTAATAATGATGG\nTAGCCCTGAAGCTGTTA\nTTAAAGAGATTAAAGA\nATGGCGAGCTGCTAAC\nGGTAAGTCAGGATTTA\nAACAGGGCTGA\n472 Lactococcin-A MKKKQIEF Lactococcus 473 ATGAAAAAAAAACAAA\nimmunity ENELRSML lactis TAGAATTTGAAAACGA\nmodulator ATALEKDI subsp. GCTAAGAAGTATGTTG\nSQEERNAL lactis GCTACCGCCCTTGAAAA\nNIAEKALD (Streptococcus AGACATTAGTCAAGAG\nNSEYLPKII lactis) GAAAGAAATGCTCTGA\nLNLRKALT ATATTGCAGAAAAGGC\nPLAINRTL GCTTGACAATTCTGAAT\nNHDLSELY ATTTACCAAAAATTATT\nKFITSSKAS TTAAACCTCAGAAAAG\nNKNLGGG CCCTAACTCCATTAGCT\nLIMSWGRLF ATAAATCGAACACTTAA\nCCATGATTTATCTGAAC\nTGTATAAATTCATTACA\nAGTTCCAAAGCATCAA\nACAAAAATTTAGGTGG\nTGGTTTAATTATGTCGT\nGGGGACGACTATTCTAA\n474 Lactococcin-A MKKKQIEF Lactococcus 475 ATGAAAAAAAAACAAA\nimmunity ENELRSML lactis TAGAATTTGAAAACGA\nmodulator ATALEKDI subsp. GCTAAGAAGTATGTTG\nSQEERNAL cremoris GCTACCGCCCTTGAAAA\nNIAEKALD (Streptococcus AGACATTAGTCAAGAG\nNSEYLPKII cremoris) GAAAGAAATGCTCTGA\nLNLRKALT ATATTGCAGAAAAGGC\nPLAINRTL GCTTGACAATTCTGAAT\nNHDLSELY ATTTACCAAAAATTATT\nKFITSSKAS TTAAACCTCAGAAAAG\nNKNLGGG CCCTAACTCCATTAGCT\nLIMSWGRLF ATAAATCGAACACTTAA\nCCATGATTTATCTGAAC\nTGTATAAATTCATTACA\nAGTTCCAAAGCATCAA\nACAAAAATTTAGGTGG\nTGGTTTAATTATGTCGT\nGGGGACGACTATTCTAA\n476 Colicin-D MNKMAMI Escherichia 477 ATGATCGATTTGGCGA\nimmunity DLAKLFLA coli AATTATTTTTAGCTTCG\nmodulator SKITAIEFS AAAATTACAGTGATTG\n(Microcin- ERICVERR AGTTTTCAGAGCGAATT\nD immunity RLYGVKDL TGTGTTGAACGGAGAA\nmodulator) SPNILNCG GATTGTATGGTGTTAAG\nEELFMAAE GATTTGTCTCCGAATAT\nRFEPDADR ATTAAATTGTGGGGAA\nANYEIDDN GAGTTGTCTATGGCTGC\nGLKVEVRS TGAGCGATTTGAGCCT\nILEKFKL GATGCAGATAGGGCTA\nATTATGAAATTGATGAT\nAATGGACTTAAGGTCG\nAGGTCCGATCTATCTTG\nGAAAAACTTAAATCAT\nAA\n478 Colicin-E5 MKLSPKAA Escherichia 479 ATGAAGTTATCACCAA\nimmunity IEVCNEAA coli AAGCTGCAATAGAAGT\nmodulator KKGLWILG TTGTAATGAAGCAGCG\n(ImmE5) IDGGHWLN AAAAAAGGCTTATGGA\n(Microcin- PGFRIDSSA TTTTGGGCATTGATGGT\nE5 SWTYDMP GGGCATTGGCTGAATC\nimmunity EEYKSKIPE CTGGATTCAGGATAGA\nmodulator) NNRLAIENI TAGTTCAGCATCATGGA\nKDDIENGY CATATGATATGCCGGA\nTAFIITLKM GAATACAAATCAAAAA\nTCCCTGAAAATAATAG\nATTGGCTATTGAAAATA\nTTAAAGATGATATTGA\nGAATGGATACACTGCTT\nTCATTATCACGTTAA\n480 Colicin-E6 MGLKLHIN Escherichia 481 ATGGGGCTTAAATTAC\nimmunity WFDKRTEE coli ATATTAATTGGTTTGAT\nmodulator FKGGEYSK AAGACGACCGAGGAAT\n(ImmE6) DFGDDGSV TTAAAGGTGGTGAGTA\n(Microcin- IERLGMPF TTCAAAAGATTTTGGAG\nE6 KDNINNG ATGATGGCTCGGTCATT\nimmunity WFDVIAEW GAACGTCTTGGAATGC\nmodulator) VPLLQPYF CTTTAAAAGATAATATC\nNHQIDISD AATAATGGTTGGTTTGA\nNEYFVSFD TGTTATAGCTGAATGG\nYRDGDW GTACCTTTGCTACAACC\nATACTTTAATCATCAAA\nTTGATATTTCCGATAAT\nGAGTATTTTGTTTCGTT\nTGATTATCGTGATGGTG\nATTGGTGA\n482 Colicin-E8 MELKKSIG Escherichia 483 GTGGAGCTAAAGAAAA\nimmunity DYTETEFK coli GTATTGGTGATTACACT\nmodulator KIIENIINCE GAAACCGAATTCAAAA\nin ColE6 GDEKKQD AAATTATTGAAAACATC\n(E8Imm[E6]) DNLEHFIS ATCAATTGTGAAGGTG\nVTEHPSGS ATGAAAAAAAACAGGA\nDLIYYPEG TGATAACCTCGAGCATT\nNNDGSPEA TTATAAGTGTTACTGAG\nVIKEIKEW CATCCTAGTGGTTCTGA\nRAANGKSG TCTGATTTATTACCCAG\nFKQG AAGGTAATAATGATGG\nTAGCCCTGAAGCTGTTA\nTTAAAGAGATTAAAGA\nATGGCGAGCTGCTAAC\nGGTAAGTCAGGATTTA\nAACAGGGCTGA\n484 Colicin-E9 MELKHSIS Escherichia 485 ATGGAACTGAAGCATA\nimmunity DYTEAEFL coli GCATTAGTGATTATACA\nmodulator QLVTTICN GAAGCTGAATTTTTACA\n(ImmE9) ADTSSEEE ACTTGTAACAACAATTT\n(Microcin- LVKLVTHF GTAATGCGAACACTTCC\nE9 EEMTEHPS AGTGAAGAAGAACTGG\nimmunity GSDLIYYP TTAAATTGGTTACACAC\nmodulator) KEGDDDSP TTTGAGGAAATGACTG\nSGIVNTVK AGCACCCTAGTGGTAG\nQWRAANG TGATTTAATATATTACC\nKSGFKQG CAAAAGAAGGTGATGA\nTGACTCACCTTCAGGTA\nTTGTAAACACAGTAAA\nACAATGGCGAGCCGCT\nAACGGTAAGTCAGGAT\nTTAAACAGGGCTAA\n486 Colicin-M MLTLYGYI Escherichia 487 ATGAAAGTAATTAGCA\nimmunity RNVFLYR coli TGAAATTTATTTTTATT\nmodulator MNDRSCG TTAACGATTATTGCTCT\n(Microcin-M DFMKVISM TGCTGCTGTTTTTTTCT\nimmunity KFIFILTIIA GGTCTGAAGATAAAGG\nmodulator) LAAVFFWS TCCGGCATGCTATCAGG\nEDKGPACY TCAGCGATGAACAGGC\nQVSDEQAR CAGAACGTTTGTAAAA\nTFVKNDYL AATGATTACCTGCAAA\nQRMKRWD GAATGAAACGCTGGGA\nNDVQLLGT CAACGATGTACAACTTC\nEIPKITWEK TTGGTACAGAAATCCC\nIERSLTDVE GAAAATTACATGGGAA\nDEKTLLVP AAGATTGAGAGAAGTT\nFKAEGPDG TAACAGATGTTGAAGA\nKRMYYGM TGAAAAAACACTTCTTG\nYHCEEGY TCCCATTTAAAGCTGAA\nVEYAND GGCCCGGACGGTAAGA\nGAATGTATTATGGCATG\nTACCATTGTGAGGAGG\nGATATGTTGAATATGCG\nAATGACTAA\n488 Colicin-B MTSNKDK Escherichia 489 ATGACCAGCAATAAAG\nimmunity NKKANEIL coli ATAAGAACAAGAAAGC\nmodulator YAFSIIGIIP AAACGAAATATTATAT\n(Microcin- LMAILILRI GCATTTTCCATAATCGG\nB immunity NDPYSQVL GATTATTCCATTAATGG\nmodulator) YYLYNKV CTATATTAATACTTCGA\nAFLPSITSL ATAAATGATCCATATTC\nHDPVMTTL TCAAGTGCTGTACTACT\nMSNYNKT TATATAATAAGGTGGC\nAPVMGILV ATTTCTCCCTTCTATTA\nFLCTYKTR CATCATTGCATGATCCC\nEIIKPVTRK GTCATGACAACACTTAT\nLVVQSCFW GTCAAACTACAACAAG\nGPVFYAILI ACAGCGCCAGTTATGG\nYITLFYNLE GTATTCTCGTTTTTCTT\nLTTAGGFF TGCACATATAAGACAA\nKLLSHNVI GAGAAATCATAAAGCC\nTLFILYCSI AGTAACAAGAAAACTT\nYFTVLTMT GTTGTGCAATCCTGTTT\nYAILLMPL CTGGGGGCCCGTTTTTT\nLVIKYFKG ATGCCATTCTGATTTAT\nRQ ATCACACTGTTCTATAA\nTCTGGAACTAACAACA\nGCAGGTGGTTTTTTTAA\nATTATTATCTCATAATG\nTCATCACTCTGTTTATT\nTTATATTGCTCCATTTA\nCTTTACTGTTTTAACCA\nTGACATATGCGATTTTA\nCTGATGCCATTACTTGT\nCATTAAATATTTTAAAG\nGGAGGCAGTAA\n490 Colicin-V MDRKRTK Escherichia 491 ATGGATAGAAAAAGAA\nimmunity LELLFAFII coli CAAAATTAGAGTTGTTA\nmodulator NATAIYIAL TTTGCATTTATAATAAA\n(Microcin- AIYDCVFR TGCCACCGCAATATATA\nV immunity GKDFLSMH TTGCATTAGCTATATAT\nmodulator) TFCFSALM GATTGTGTTTTTAGAGG\nSAICYFVG AAAGGACTTTTTATCCA\nDNYYSISD TGCATACATTTTGCTTC\nKIKRRSYE TCTGCATTAATGTCTGC\nNSDSK AATATGTTACTTTGTTG\nGTGATAATTATTATTCA\nATATCCGATAAGATAA\nAAAGGAGATCATATGA\nGAACTCTGACTCTAAAT\nGA\n492 Colicin- MSLRYYIK Shigella 493 ATGAGTTTAAGATACTA\nE1* NILFGLYC sonnei CATAAAAAATATTTTGT\nimmunity ALIYIYLIT TTGGCCTATACTGCGCA\nmodulator KNNEGYYF CTTATATATATATACCT\n(ImmE1) LASDKMLY TATAACAAAAAACAAC\n(Microcin- AIVISTILCP GAAGGGTATTATTTCCT\nE1* YSKYAIEHI AGCGTCAGATAAGATG\nimmunity FFKFIKKDF CTATACGCAATAGTGAT\nmodulator) FRKRKNLN AAGCACTATTCTATGCC\nKCPRGKIK CATATTCAAAATATGCT\nPYLCVYNL ATTGAACACATATTTTT\nLCLVLAIPF TAAGTTCATAAAGAAA\nGLLGLVYI GATTTTTTCAGAAAAAG\nNKE AAAAAACCTAAATAAA\nTGCCCCCGTGGCAAAA\nTTAAACCGTATTTATGC\nGTATACAATCTACTTTG\nTTTGGTCCTAGCAATCC\nCATTTGGATTGCTAGGA\nCTTGTTTATATCAATAA\nAGAATAA\n494 Colicin-E1 MSLRYYIK Escherichia 495 ATGAGCTTAAGATACTA\nimmunity NILFGLYC coli CATAAAAAATATTTTAT\nmodulator TLIYIYLIT TTGGCCTGTACTGCACA\n(ImmE1) KNSEEYYF CTTATATATATATACCT\n(Microcin- LVTDKML TATAACAAAAAACAGC\nE1 YAIVISTIL GAAGAGTATTATTTCCT\nimmunity CPYSKYAI TGTGACAGATAAGATG\nmodulator) EHIAFNFIK CTATATGCAATAGTGAT\nKHFFERRK AAGCACTATTCTATGTC\nNLNNAPVA CATATTCAAAATATGCT\nKLNLFMLY ATTGAACACATAGCTTT\nNLLCLVLA TAACTTCATAAAGAAAC\nIPFGLLGLF ATTTTTTCGAAAGAAGA\nISIKNN AAAAACCTAAATAACG\nCCCCCGTAGCAAAATTA\nAACCTATTTATGCTATA\nTAATCTACTTTGTTTGG\nTCCTAGCAATCCCATTT\nGGATTGCTAGGACTTTT\nTATATCAATAAAGAATA\nATTAA\n496 Probable MRKNNILL Leuconostoc 497 TTGAGAAAAAATAACA\nleucocin-A DDAKIYTN gelidum TTTTATTGGACGATGCT\nimmunity KLYLLLID AAAATATACACGAACA\nmodulator RKDDAGY AACTCTATTTGCTATTA\nGDICDVLF ATCGATAGAAAAGATG\nQVSKKLDS ACGCTGGGTATGGAGA\nTKNVEALI TATTTGTGATGTTTTGT\nNRLVNYIRI TTCAGGTATCCAAAAA\nTASTNRIKF ATTAGATAGCACAAAA\nSKDEEAVII AATGTAGAAGCATTGA\nELGVIGQK TTAACCGATTGGTCAAT\nAGLNGQY TATATACGAATTACCGC\nMADFSDKS TTCAACAAACAGAATTA\nQFYSIFER AGTTTTCAAAAGATGA\nAGAGGCTGTAATTATA\nGAACTTGGTGTAATTG\nGTCAGAAGGCTGGATT\nAAACGGCCAATACATG\nGCTGATTTTTCTGACAA\nATCTCAGTTTTATAGTA\nTCTTTGAAAGATAA\n498 Lactococcin-B MKKKVDT Lactococcus 499 ATGAAAAAAAAAGTTG\nimmunity EKQITSWA lactis ATACAGAAAAACAAAT\nmodulator SDLASKNE subsp. TACTTCTTGGGCATCTG\nTKVQEKLI cremoris ACTTAGCTTCCAAAAAT\nLSSYIQDIE (Streptococcus GAAACAAAGGTTCAAG\nNHVYFPKA cremoris) AAAAATTAATACTGTCT\nMISLEKKL TCTTATATTCAGGACAT\nRDQNNICA CGAAAACCATGTTTACT\nLSKEVNQF TTCCAAAAGCAATGATT\nYFKVVEVN TCTTTAGAAAAAAAATT\nQRKSWMV ACGAGACCAAAATAAT\nGLIV ATTTGCGCTTTATCAAA\nAGAAGTCAATCAGTTTT\nATTTTAAAGTTGTTGAA\nGTAAATCAAAGAAAAT\nCCTGGATGGTAGGTTTG\nATAGTTTAA\n500 Pediocin MNKTKSE Pediococcus 501 ATGAATAAGACTAAGT\nPA-1 HIKQQALD acidilactici CGGAACATATTAAACA\nimmunity LFTRLQFLL ACAAGCTTTGGACTTAT\nmodulator QKHDTIEP TTACTAGGCTACAGTTT\n(Pediocin YQYVLDIL TTACTACAGAAGCACG\nACH ETGISKTK ATACTATCGAACCTTAC\nimmunity HNQQTPER CAGTACGTTTTAGATAT\nmodulator) QARVVYN TCTGGAGACTGGTATCA\nKIASQALV GTAAAACTAAACATAA\nDKLHFTAE CCAGCAAACGCCTGAA\nENKVLAAI CGACAAGCTCGTGTAG\nNELAHSQK TCTACAACAAGATTGCC\nGWGEFNM AGCCAAGCGTTAGTAG\nLDTTNTWP ATAAGTTACATTTTACT\nSQ GCCGAAGAAAACAAAG\nTTCTAGCAGCCATCAAT\nGAATTGGCGCATTCTCA\nAAAAGGGTGGGGCGAG\nTTTAACATGCTAGATAC\nTACCAATACGTGGCCTA\nGCCAATAG\n502 Putative MIKDEKIN Carnobacterium 503 ATGATAAAAGATGAAA\ncarnobacteriocin- KIYALVKS maltaromaticum AAATAAATAAAATCTAT\nBM1 ALDNTDV (Carnobacterium GCTTTAGTTAAGAGCGC\nimmunity KNDKKLSL piscicola) ACTTGATAATACGGAT\nmodulator LLMRIQET GTTAAGAATGATAAAA\nSINGELFY AACTTTCTTTACTTCTT\nDYKKELQP ATGAGAATACAAGAAA\nAISMYSIQ CATCAATTAATGGAGA\nHNFRVPDD ACTATTTTACGATTATA\nLVKLLALV AAAAAGAATTACAGCC\nQTPKAWS AGCTATTAGTATGTACT\nGF CTATTCAACATAACTTT\nCGGGTTCCTGACGATCT\nAGTAAAACTGTTAGCAT\nTAGTTCAAACACCTAAA\nGCTTGGTCAGGGTTTTAA\n504 Putative MDIKSQTL Carnobacterium 505 ATGGATATAAAGTCTCA\ncarnobacteriocin- YLNLSEAY maltaromaticum AACATTATATTTGAATC\nB2 KDPEVKAN (Carnobacterium TAAGCGAGGCATATAA\nimmunity EFLSKLVV piscicola) AGACCCTGAAGTAAAA\nmodulator QCAGKLTA GCTAATGAATTCTTATC\n(Carnocin- SNSENSYIE AAAATTAGTTGTACAAT\nCP52 VISLLSRGI GTGCTGGGAAATTAAC\nimmunity SSYYLSHK AGCTTCAAACAGTGAG\nmodulator) RIIPSSMLTI AACAGTTATATTGAAGT\nYTQIQKDI AATATCATTGCTATCTA\nKNGNIDTE GGGGTATTTCTAGTTAT\nKLRKYEIA TATTTATCCCATAAACG\nKGLMSVPY TATAATTCCTTCAAGTA\nIYF TGTTAACTATATATACT\nCAAATACAAAAGGATA\nTAAAAAACGGGAATAT\nTGACACCGAAAAATTA\nAGGAAATATGAGATAG\nCAAAAGGATTAATGTC\nCGTTCCTTATATATATT\nTCTAA\n506 Nisin MRRYLILI Lactococcus 507 ATGAGAAGATATTTAAT\nimmunity VALIGITGL lactis ACTTATTGTGGCCTTAA\nmodulator SGCYQTSH subsp. TAGGGATAACAGGTTT\nKKVRFDEG lactis ATCAGGGTGTTATCAA\nSYTNFIYD (Streptococcus ACAAGTCATAAAAAGG\nNKSYFVTD lactis) TGAGGTTTGACGAAGG\nKEIPQENV AAGTTATACTAATTTTA\nNNSKVKFY TTTATGATAATAAATCG\nKLLIVDMK TATTTCGTAACTGATAA\nSEKLLSSSN GGAGATTCCTCAGGAG\nKNSVTLVL AACGTTAACAATTCCAA\nNNIYEASD AGTAAAATTTTATAAGC\nKSLCMGIN TGTTGATTGTTGACATG\nDRYYKILP AAAAGTGAGAAACTTT\nESDKGAVK TATCAAGTAGCAACAA\nALRLQNFD AAATAGTGTGACTTTGG\nVTSDISDD TCTTAAATAATATTTAT\nNFVIDKND GAGGCTTCTGACAAGT\nSRKIDYMG CGCTATGTATGGGTATT\nNIYSISDTT AACGACAGATACTATA\nVSDEELGE AGATACTTCCAGAAAG\nYQDVLAE TGATAAGGGGGCGGTC\nVRVFDSVS AAAGCTTTGAGATTACA\nGKSIPRSE AAACTTTGATGTGACAA\nWGRIDKD GCGATATTTCTGATGAT\nGSNSKQSR AATTTTGTTATTGATAA\nTEWDYGEI AAATGATTCACGAAAA\nHSIRGKSLT ATTGACTATATGGGAA\nEAFAVEIN ATATTTACAGTATATCG\nDDFKLATK GACACCACCGTATCTGA\nVGN TGAAGAATTGGGAGAA\nTATCAGGATGTTTTAGC\nTGAAGTACGTGTGTTTG\nATTCAGTTAGTGGCAA\nAAGTATCCCGAGGTCT\nGAATGGGGGAGAATTG\nATAAGGATGGTTCAAA\nTTCCAAACAGAGTAGG\nACGGAATGGGATTATG\nGCGAAATCCATTCTATT\nAGAGGAAAATCTCTTA\nCTGAAGCATTTGCCGTT\nGAGATAAATGATGATT\nTTAAGCTTGCAACGAA\nGGTAGGAAACTAG\n508 Trifolitoxin MNDEICLT Rhizobium 509 ATGAATGATGAGATTT\nimmunity GGGRTTVT leguminosarum GCCTGACAGGTGGCGG\nmodulator RRGGVVY bv. ACGAACGACTGTCACG\nREGGPWSS trifolii CGGCGCGGCGGAGTCG\nTVISLLRHL TGTATCGCGAAGGCGG\nEASGFAEA CCCGTGGTCATCAACCG\nPSVVGTGF TCATTTCGCTCCTGCGG\nDERGRETL CATCTGGAAGCCTCTGG\nSFIEGEFVH CTTCGCTGAAGCTCCTT\nPGPWSEEA CCGTTGTCGGCACCGGT\nFPQFGMML TTCGATGAGCGCGGCC\nRRLHDATA GGGAGACATTATCGTTT\nSFKPPENS ATCGAGGGTGAGTTTG\nMWRDWFG TTCACCCAGGCCCTTGG\nRNLGEGQH TCGGAGGAGGCTTTTCC\nVIGHCDTG GCAATTTGGAATGATGT\nPWNIVCRS TGCGGCGACTGCACGA\nGLPVGLID TGCCACCGCCTCGTTCA\nWEVAGPV AACCTCCCGAAAACTC\nRADIELAQ GATGTGGCGCGATTGG\nACWLNAQ TTCGGGCGTAACCTCG\nLYDDDIAE GTGAGGGTCAACACGT\nRVGLGSVT AATAGGACACTGCGAC\nMRAHQVR ACAGGCCCATGGAACA\nLLLDGYGL TTGTTTGCCGGTCAGGA\nSRKQRGGF TTGCCTGTCGGGTTGAT\nVDKLITFA AGATTGGGAGGTGGCT\nVHDAAEQ GGGCCTGTCAGGGCGG\nAKEAAVTP ATATCGAATTGGCCCA\nESNDAEPL GGCTTGTTGGCTGAATG\nWAIAWRT CCCAGCTCTACGATGAC\nRSASWML GACATTGCGGAGAGGG\nHHRQTLEA TCGGATTAGGCTCTGTG\nALA ACCATGAGAGCGCATC\nAAGTTCGCCTGCTGCTT\nGACGGCTATGGTCTGTC\nTCGGAAGCAACGCGGC\nGGCTTCGTCGACAAGCT\nAATCACGTTCGCAGTTC\nACGATGCGGCCGAGCA\nGGCGAAAGAGGCGGCT\nGTCACGCCAGAGTCGA\nACGATGCGGAACCGCT\nATGGGCAATTGCCTGG\nCGCACTAGAAGTGCCT\nCCTGGATGCTCCATCAT\nCGGCAAACACTGGAAG\nCAGCGCTGGCATAG\n510 Antilisterial MNNIIPIMS Bacillus 511 ATGAATAACATAATCCC\nbacteriocin LLFKQLYS subtilis TATCATGTCTTTGCTGT\nsubtilosin RQGKKDAI (strain 168) TCAAACAGCTTTACAGC\nbiosynthesis RIAAGLVIL CGGCAAGGGAAAAAGG\nprotein AVFEIGLIR ACGCCATCCGCATTGCC\nAlbD QAGIDESV GCAGGCCTTGTCATTCT\nLRKTYIILA GGCCGTGTTTGAAATC\nLLLMNTY GGGCTGATCCGCCAGG\nMVFLSVTS CCGGCATTGATGAATC\nQWKESYM GGTGTTGCGCAAAACG\nKLSCLLPIS TATATCATACTCGCGCT\nSRSFWLAQ TCTTTTGATGAACACAT\nSVVLFVDT ATATGGTGTTTCTTTCC\nCLRRTLFFF GTGACATCACAATGGA\nILPLFLFGN AGGAATCTTATATGAA\nGTLSGAQT GCTGAGCTGCCTGCTGC\nLFWLGRFS CGATTTCTTCACGGAGC\nFFTVYSIIF TTTTGGCTCGCCCAGAG\nGVVLSNHF TGTCGTTTTGTTTGTCG\nVKKKNLM ATACCTGTTTGAGAAG\nFLLHAAIFA AACTTTATTCTTTTTTA\nCVCISAAL TTTTACCGCTGTTCTTA\nMPAATIPL TTTGGAAACGGAACGC\nCAVHILWA TGTCAGGGGCGCAAAC\nVVIDFPVFL ATTGTTTTGGCTCGGCA\nQAPPQQGK GGTTTTCGTTTTTTACC\nMHSFMRRS GTTTACTCCATTATTTT\nEFSFYKRE CGGAGTTGTGCTAAGC\nWNRFISSK AACCACTTCGTCAAAAA\nAMLLNYA GAAGAACTTGATGTTTC\nVMAVFSGF TGCTGCATGCGGCGAT\nFSFQMMNT ATTCGCCTGTGTATGTA\nGIFNQQVI TCAGCGCCGCTTTGATG\nYIVISALLL CCGGCCGCCACGATTCC\nICSPIALLY GCTTTGCGCGGTTCATA\nSIEKNDRM TCCTGTGGGCGGTGGT\nLLITLPIKR CATTGACTTTCCTGTCT\nKTMFWAK TTCTGCAGGCGCCTCCG\nYRFYSGLL CAGCAGGGCAAGATGC\nAGGFLLVV ATTCATTTATGCGGCGA\nMIVGFISGR TCTGAATTTTCGTTTTA\nSISVLTFLQ CAAAAGAGAATGGAAC\nCIELLLAG CGATTTATCTCTTCTAA\nAYIRLTAD AGCGATGCTGTTAAATT\nEKRPSFSW ACGCGGTAATGGCGGT\nQTEQQLWS ATTCAGCGGCTTCTTTT\nGFSKYRSY CGTTCCAGATGATGAA\nLFCLPLFLA CACCGGCATCTTCAATC\nILAGTAVS AGCAAGTGATTTATATC\nLAVIPIAGL GTGATTTCCGCGCTTTT\nVIVYYLQK GCTCATCTGCTCGCCGA\nQDGGFFDT TCGCCCTTTTGTATTCG\nSKRERLGS ATTGAAAAAAATGACC\nGGATGCTGCTCATCACG\nCTTCCGATCAAGCGAA\nAAACGATGTTTTGGGC\nGAAATATCGCTTTTATT\nCAGGCCTATTGGCAGG\nCGGATTTCTCCTTGTCG\nTGATGATTGTGGGTTTCA\n512 Putative MSILDIHD Bacillus 513 GCATTTTGGATATACAC\nABC VSVWYER subtilis GATGTATCCGTTTGGTA\ntransporter DNVILEQV (strain 168) TGAACGGGACAACGTC\nATP- DLHLEKGA ATCTTAGAGCACGTGG\nbinding VYGLLGV ACTTACACTTAGAAAAA\nprotein NGAGKTTL GGCGCCGTTTACGGATT\nAlbC INTLTGVN GCTTGGGGTAAACGGT\n(Antilisterial RNFSGRFT GCCGGCAAAACAACAC\nbacteriocin LCGIEAEA TGATCAATACGCTGACA\nsubtilosin GMPQKTSD GGAGTGAACCGCAATT\nbiosynthesis QLKTHRYF ACAGCGGGGGCTTTAC\nprotein AADYPLLF GCTGTGCGGCATTGAA\nAlbC) TEITAKDY GCTGAGGCCGGCATGC\nVSFVHSLY CGCAGAAAACATCAGA\nQKDFSEQQ TCAACTGAAGATTCACC\nFASLAEAF GTTACTTCGCCGCTGAT\nHFSKYINR TATCCGCTGCTGTTTAC\nRISELSLGN AGAAATTACGGCGAAG\nRQKVVLM GACTATGTGTCTTTCGT\nTGLLLRAP CCATTCGCTTTATCAAA\nLFILDEPLV AGGATTTTTCAGAGCG\nGLDVESIE ACAGTTTGCCAGTTTGG\nVFYQKMR CTGAGGCCTTTCATTTT\nEYCEAGGT TCAAAATACATCAACA\nILFSSHLLD GGAGAATCTCGGAGCT\nVVQRFCDY GTCCTTGGGGAACAGG\nAAILHNKQ CAAAAGGTTGTGTTGAT\nIQKVIPIGE GACAGGATTATTGCTGC\nETDLRREF GGGCTCCCCTGTTTATT\nFEVIGHE TTGGATGAGCCGCTCGT\nCGGTTTGGATGTGGAA\nTCAATAGAGGTCTTTTA\nTCAGAAAATGCGGGAG\nTACTGTGAGGAAGGCG\nGAACCATTTTGTTTTCT\nTCCCATCTGCTCGATGT\nCGTGCAGAGATTTTGTG\nATTTTGCGGCCATTCTG\nCACAACAAACAGATCC\nAAAAGGTCATTCCGATT\nGGGGAGGAGACCGATC\nTGCGGCGGGAATTTTTT\nGAGGTTATCGGCCATG\nAATAA\n514 Antilisterial MSPAQRRI Bacillus 515 TTGTCACCAGCACAAA\nbacteriocin LLYILSFIF subtilis GAAGAATTTTACTGTAT\nsubtilosin VIGAVVYF (strain 168) ATCCTTTCATTTATCTT\nbiosynthesis VKSDYLFT TGTCATCGGCGCAGTC\nprotein LIFIAIAILF GTCTATTTTGTCAAAAG\nAlbB GMRARKA CGATTATCTGTTTACGC\nDSR TGATTTTCATTGCCATT\nGCCATTCTGTTCGGGAT\nGCGCGCGCGGAAGGCT\nGACTCGCGATGA\n516 Colicin-E7 MELKNSIS Escherichia 517 ATGGAACTGAAAAATA\nimmunity DYTEAEFV coli GTATTAGTGATTACACA\nmodulator QLLKEIEK GAGGCTGAGTTTGTTCA\n(ImmE7) ENVAATD ACTTCTTAAGGAAATTG\n(Microcin- DVLDVLLE AAAAAGAGAATGTTGC\nE7 HFVKITEH TGCAACTGATGATGTGT\nimmunity PDGTDLIY TAGATGTGTTACTCGAA\nmodulator) YPSDNRDD CACTTTGTAAAAATTAC\nSPEGIVKEI TGAGCATCCAGATGGA\nKEWRAAN ACGGATCTGATTTATTA\nGKPGFKQG TCCTAGTGATAATAGA\nGACGATAGCCCCGAAG\nGGATTGTCAAGGAAAT\nTAAAGAATGGCGAGCT\nGCTAACGGTAAGCCAG\nGATTTAAACAGGGCTGA\n518 Pyocin-S1 MKSKISEY Pseudomonas 519 ATGAAGTCCAAGATTTC\nimmunity TEKEFLEF aeruginosa CGAATATACGGAAAAA\nmodulator VEDIYTNN GAGTTTCTTGAGTTTGT\nKKKFPTEE TGAAGACATATACACA\nSHIQAVLE AACAATAAGAAAAAGT\nFKKLTEHP TCCCTACCGAGGAGTCT\nSGSDLLYY CATATTCAAGCCGTGCT\nPNENREDS TGAATTTAAAAAACTAA\nPAGVVKEV CGGAACACCCAAGCGG\nKEWRASK CTCAGACCTTCTTTACT\nGLPGFKAG ACCCCAACGAAAATAG\nAGAAGATAGCCCAGCT\nGGAGTTGTAAAGGAAG\nTTAAAGAATGGCGTGC\nTTCCAAGGGGCTTCCTG\nGCTTTAAGGCCGGTTAG\n520 Pyocin-S2 MKSKISEY Pseudomonas 521 ATGAAGTCCAAGATTTC\nimmunity TEKEFLEF aeruginosa CGAATATACGGAAAAA\nmodulator VKDIYTNN (strain GAGTTTCTTGAGTTTGT\nKKKFPTEE ATCC TAAAGACATATACACA\nSHIQAVLE 15692/ AACAATAAGAAAAAGT\nFKKLTEHP PAO1/1C/ TCCCTACCGAGGAGTCT\nSGSDLLYY PRS 101/ CATATTCAAGCCGTGCT\nPNENREDS LMG TGAATTTAAAAAACTAA\nPAGVVKEV 12228) CGGAACACCCAAGCGG\nKEWRASK CTCAGACCTTCTTTACT\nGLPGFKAG ACCCCAACGAAAATAG\nAGAAGATAGCCCAGCT\nGGAGTTGTAAAGGAAG\nTTAAAGAATGGCGTGC\nTTCCAAGGGGCTTCCTG\nGCTTTAAGGCCGGTTAG\n522 Hiracin- MDFTKEEK Enterococcus 523 ATGGATTTTACTAAAGA\nJM79 LLNAISKV hirae AGAAAAACTTTTAAAT\nimmunity YNEATIDD GCAATTAGTAAAGTAT\nfactor YPDLKEKL ACAATGAAGCAACTAT\nFLYSKEISE AGATGACTATCCTGACT\nGKSVGEVS TAAAAGAAAAGCTCTTT\nMKLSSFLG CTTTATTCTAAAGAAAT\nRYILKHKF CAGTGAGGGAAAAAGT\nGLPKSLIEL GTTGGTGAAGTTAGTAT\nQEIVSKES GAAATTAAGTAGTTTTC\nQVYRGWA TTGGAAGATATATTTTA\nSIGIWS AAACATAAATTTGGATT\nACCTAAATCTTTAATAG\nAATTACAAGAAATTGTT\nAGTAAGGAATCTCAAG\nTATATAGAGGATGGGC\nTTCTATTGGTATTTGGA\nGTTAA\n524 Probable MKKKYRY Leuconostoc 525 TTGAAAAAAAAGTATC\nmesentericin- LEDSKNYT mesenteroides GGTATTTAGAAGATAG\nY105 STLYSLLV CAAAAATTACACTAGTA\nimmunity DNVDKPG CACTCTATTCTCTGTTA\nmodulator YSDICDVL GTTGATAATGTTGACAA\nLQVSKKLD ACCTGGATACTCAGATA\nNTQSVEAL TTTGCGATGTTTTGCTT\nINRLVNYIR CAAGTTTCTAAGAAGTT\nITASTYKIIF GGATAATACTCAAAGT\nSKKEEELII GTTGAAGCGCTAATTA\nKLGVIGQK ATCGATTGGTTAATTAT\nAGLNGQY ATTCGTATTACTGCTTC\nMADFSDKS AACATACAAAATTATTT\nQFYSVFDQ TTTCAAAAAAAGAAGA\nGGAATTGATTATAAAA\nCTTGGTGTTATTGGACA\nAAAAGCTGGACTTAAT\nGGTCAGTATATGGCTG\nATTTTTCAGACAAGTCT\nCAGTTTTACAGCGTTTT\nCGATCAGTAA\n526 Microcin- MSFLNFAF Escherichia 527 ATGAGTTTTCTTAATTT\n24 SPVFFSIMA coli TGCATTTTCTCCTGTAT\nimmunity CYFIVWRN TCTTCTCCATTATGGCG\nmodulator KRNEFVCN TGTTATTTCATTGTATG\nRLLSIIIISFL GAGAAATAAACGAAAC\nICFIYPWLN GAATTTGTCTGCAATAG\nYKIEVKYY ATTGCTATCAATTATAA\nIFEQFYLFC TAATATCTTTTTTGATA\nFLSSLVAV TGCTTCATATATCCATG\nVINLIVYFI GCTAAATTACAAAATC\nLYRRCI GAAGTTAAATATTATAT\nATTTGAACAGTTTTATC\nTTTTTTGTTTTTTATCGT\nCACTCGTGGCTGTTGTA\nATAAACCTAATTGTATA\nCTTTATATTATACAGGA\nGATGTATATGA\n528 Colicin-K MHLKYYL Escherichia 529 ATGCATTTAAAATACTA\nimmunity HNLPESLIP coli CCTACATAATTTACCTG\nmodulator WILILIFND AATCACTTATACCATGG\nNDNTPLLFI ATTCTTATTTTAATATT\nFISSIHVLL TAACGACAATGATAAC\nYPYSKLTIS ACTCCTTTGTTATTTAT\nRYIKENTK ATTTATATCATCAATAC\nLKKEPWYL ATGTATTGCTATATCCA\nCKLSALFY TACTCTAAATTAACCAT\nLLMAIPVG ATCTAGATATATCAAAG\nLPSFIYYTL AAAATACAAAGTTAAA\nKRN AAAAGAACCCTGGTAC\nTTATGCAAGTTATCTGC\nATTGTTTTATTTATTAA\nTGGCAATCCCAGTAGG\nATTGCCAAGTTTCATAT\nATTACACTCTAAAGAG\nAAATTAA\n530 Microcin MMIQSHPL Escherichia 531 ATGATGATACAATCTCA\nC7 self- LAAPLAVG coli TCCACTACTGGCCGCTC\nimmunity DTIGFFSSS CCCTGGCAGTAGGAGA\nmodulator APATVTAK TACAATTGGTTTCTTTT\nMccF NRFFRGVE CATCATCTGCTCCGGCA\nFLQRKGFK ACAGTTACTGCAAAAA\nLVSGKLTG ATCGTTTTTTTCGGGGA\nKTDFYRSG GTTGAGTTTCTTCAGAG\nTIKERAQE AAAGGGATTTAAGCTG\nFNELVYNP GTATCAGGGAAGCTTA\nDITCIMSTI CCGGTAAAACAGATTTT\nGGDNSNSL TATCGTTCAGGTACTAT\nLPFLDYDA TAAAGAAAGAGCTCAA\nIIANPKIIIG GAATTTAATGAGTTAGT\nYSDTTALL CTACAATCCTGATATTA\nAGIYAKTG CCTGTATAATGTCAACG\nLITFYGPAL ATCGGTGGAGATAACA\nIPSFGEHPP GTAATTCACTACTACCG\nLVDITYESF TTTCTGGACTATGATGC\nIKILTRKQS TATCATTGCAAACCCCA\nGIYTYTLP AAATTATCATAGGTTAC\nEKWSDESI TCAGATACAACTGCTTT\nNWNENKIL ATTAGCAGGAATATAT\nRPKKLYKN GCAAAAACAGGGTTAA\nNCAFYGSG TAACATTCTATGGACCA\nKVEGRVIG GCTCTTATTCCTTCGTT\nGNLNTLTG TGGTGAACATCCACCTC\nIWGSEWM TTGTGGATATAACATAT\nPEILNGDIL GAATCATTTATTAAAAT\nFIEDSRKSI ACTAACAAGAAAACAA\nATIERLFS TCAGGAATATATACCTA\nMLKLNRVF CACATTACCTGAAAAGT\nDKVSAIILG GGAGTGATGAGAGCAT\nKHELFDCA AAACTGGAATGAAAAC\nGSKRRPYE AAGATATTAAGGCCTA\nVLTEVLDG AGAAGCTATATAAAAA\nKQIPVLDG CAACTGTGCCTTTTATG\nFDCSHTHP GTTCCGGAAAAGTTGA\nMLTLPLGV GGGGCGTGTAATTGGA\nKLAIDFDN GGAAATCTAAATACTTT\nKNISITEQY GACAGGTATATGGGGG\nLSTEK AGTGAATGGATGCCTG\nAAATTCTTAATGGAGAT\nATATTGTTTATTGAGGA\nCAGTCGGAAAAGCATT\nGCAACAATTGAACGAT\nTATTCTCTATGCTAAAG\nCTTAATCGCGTGTTTGA\nTAAAGTTAGTGCAATA\nATACTCGGGAAACATG\nAGCTTTTTGATTGTGCA\nGGAAGTAAACGCAGAC\nCATATGAAGTATTAACA\nGAGGTATTAGATGGGA\nAACAGATTCCTGTACTG\nGATGGATTTGATTGTTC\nACATACACATCCAATGC\nTAACTCTTCCACTTGGT\nGTAAAATTAGCTATTGA\nCTTTGACAACAAAAATA\nTAT\n532 Sakacin-A MKADYKKI Lactobacillus 533 GGCAGATTATAAAAAA\nimmunity NSILTYTST sakei ATAAATTCAATACTAAC\nfactor ALKNPKIIK TTACACATCTACTGCTT\nDKDLVVLL TAAAAAACCCTAAAATT\nTIIQEEAKQ ATAAAAGATAAAGATT\nNRIFYDYK TAGTAGTCCTTCTAACT\nRKFRPAVT ATTATTCAAGAAGAAG\nRFTIDNNFE CCAAACAAAATAGAAT\nIPDCLVKL CTTTTATGATTATAAAA\nLSAVETPK GAAAATTTCGTCCAGC\nAWSGFS GGTTACTCGCTTTACAA\nTTGATAATAATTTTGAG\nATTCCTGATTGTTTGGT\nTAAACTACTGTCAGCTG\nTTGAAACACCTAAGGC\nGTGGTCTGGATTTAGTT\nAG\n534 Colicin-E5 MKLSPKAA Escherichia 535 TGAAGTTATCACCAAA\nimmunity IEVCNEAA coli AGCTGCAATAGAAGTT\nmodulator KKGLWILG TGTAATGAAGCAGCGA\nin ColE9 IDGGHWLN AAAAAGGCTTATGGAT\n(E5Imm[E9]) PGFRIDSSA TTTGGGCATTGATGGTG\nSWTYDMP GGCATTGGCTGAATCCT\nEEYKSKTP GGATTCAGGATAGATA\nENNRLAIE GTTCAGCATCATGGAC\nNIKDDIEN ATATGATATGCCGGAG\nGYTAFIITL GAATACAAATCAAAAA\nKM CCCCTGAAAATAATAG\nATTGGCTATTGAAAATA\nTTAAAGATGATATTGA\nGAATGGATACACTGCTT\nTCATTATCACGTTAAAG\nATGTAA\n536 Antilisterial MNNIFPIM Bacillus 537 TTGGGGAGGAGACCGA\nbacteriocin SLLFKQLY subtilis TCTGCGGCGGGAATTTT\nsubtilosin SRQGKKDA TTGAGGTTATCGGCCAT\nbiosynthesis IRIAAGLVI GAATAACATATTCCCCA\nprotein LAVFEIGLI TCATGTCGTTGCTGTTC\nAlbD RQAGIDES AAACAGCTGTACAGCC\nVLGKTYIIL GGCAAGGGAAAAAGGA\nALLLMNTY CGCTATCCGCATTGCTG\nMVFLSVTS CAGGGCTTGTGATTCTC\nQWKESYM GCCGTGTTTGAAATCG\nKLSCLLPIS GGCTGATCCGACAAGC\nSRSFWLAQ CGGCATTGACGAATCG\nSVVLFVDT GTGTTGGGAAAAACGT\nCLRRTLFFF ATATCATATTGGCGCTT\nILPLFLFGN CTCTTAATGAACACGTA\nGTLSGAQT TATGGTGTTTCTTTCCG\nLFWLGRFS TGACATCACAATGGAA\nFFTVYSILF GGAATCTTATATGAAG\nGVMLSNHF CTGAGCTGTCTGCTGCC\nVKKKNSM GATTTCATCACGGAGCT\nFLLHAAVF TTTGGCTCGCCCAGAGT\nAFVCLSAA GTCGTTCTGTTTGTCGA\nFMPAVTIP TACCTGTTTGAGAAGA\nLCAVHML ACGTTATTCTTTTTTAT\nWAVIIDFP TTTACCGCTGTTCTTAT\nVFLQAPPH TTGGAAACGGAACGCT\nQSKMHFF GTCAGGGGCGCAAACA\nMRRSEFSF TTGTTTTGGCTTGGCAG\nYKREWNR ATTTTCGTTTTTTACCG\nFISSKAMLL TTTACTCGATTCTATTC\nNYVVMAA GGAGTTATGCTAAGCA\nFSGFFSFQ ACCATTTCGTCAAAAAG\nMMNTGIFN AAGAACTCGATGTTTCT\nQQVIYIVIS GCTGCATGCGGCGGTA\nALLLICSPI TTCGCCTTTGTATGCCT\nALLYSIEK CAGTGCCGCTTTTATGC\nNDRMLLIT CGGCCGTCACGATCCC\nLPIKRRTM GCTATGCGCGGTTCACA\nFWAKYRF TGCTATGGGCGGTGAT\nYSGLLAGG CATTGACTTTCCGGTCT\nFLLVAIIVG TTCTGCAGGCGCCTCCG\nFISGRPISA CATCAGAGCAAGATGC\nLTFVQCME ATTTTTTTATGCGGCGA\nLLLAGAFIR TCTGAATTTTCGTTTTA\nLTADEKRP CAAAAGAGAATGGAAC\nSFGWQTEQ CGATTTATTTCTTCTAA\nQLWSGFSK AGCGATGCTGTTAAATT\nYRSYLFCL ACGTGGTGATGGCGGC\nPLFLATLA GTTCAGCGGATTCTTTT\nGTAVSLAV CGTTCCAGATGATGAA\nIPIAALIIVY CACTGGCATCTTCAATC\nYLQKQDG AGCAAGTGATTTATATT\nGFFDTSKR GTGATTTCCGCTCTATT\nERIGS GCTGATTTGCTCGCCGA\nTCGCCCTTTTGTACTCT\nATTGAAAAAAACGATC\nGCATGCTGCTCATCACG\nCTTCCAATTAAAAGAA\nGAACGATGTTTTGGGC\nGAAATATCGCTTTTATT\nCAG\n538 Microcin- MERKQKN Escherichia 539 ATGGAAAGAAAACAGA\nJ25 export SLFNYIYSL coli AAAACTCATTATTTAAT\nATP- MDVRGKF TATATTTATTCATTAAT\nbinding/permease LFFSMLFIT GGATGTAAGAGGTAAA\nprotein SLSSIIISISP TTTTTATTCTTTTCCAT\nMcjD LILAKITDL GTTATTCATTACATCAT\n(Microcin- LSGSLSNFS TATCATCGATAATCATA\nJ25 YEYLVLLA TCTATTTCACCATTGAT\nimmunity CLYMFCVI TCTTGCAAAGATTACAG\nmodulator) SNKASVFL ATTTACTGTCTGGCTCA\n(Microcin- FMILQSSLR TTGTCAAATTTTAGTTA\nJ25 INMQKKM TGAATATCTGGTTTTAC\nsecretion SLKYLREL TTGCCTGTTTATACATG\nATP- YNENITNL TTTTGCGTTATATCTAA\nbinding SKNNAGYT TAAAGCAAGTGTTTTTT\nprotein TQSLNQAS TATTTATGATACTGCAA\nMcjD) NDIYILVR AGTAGTCTACGTATTAA\nNVSQNILS CATGCAGAAAAAAATG\nPVIQLISTI TCGCTAAAGTATTTGAG\nVVVLSTKD AGAATTGTATAACGAA\nWFSAGVFF AATATAACTAACTTGAG\nLYILVFVIF TAAAAATAATGCTGGA\nNTRLTGSL TATACAACGCAAAGTCT\nASLRKHSM TAACCAGGCTTCAAATG\nDITLNSYSL ACATTTATATTCTTGTG\nLSDTVDN AGAAATGTTTCCCAGA\nMIAAKKNN ATATCCTGTCACCTGTT\nALRLISERY ATACAACTTATTTCCAC\nEDALTQEN TATTGTTGTTGTTTTAT\nNAQKKYW CTACGAAGGACTGGTTT\nLLSSKVLL TCTGCCGGTGTGTTTTT\nLNSLLAVIL TCTCTATATTCTGGTAT\nFGSVFIYNI TTGTAATTTTTAATACC\nLGVLNGV AGACTGACTGGCAGTTT\nVSIGHFIMI AGCGTCTCTCAGAAAA\nTSYIILLST CACAGCATGGATATCA\nPVENIGAL CTCTTAACTCTTATAGT\nLSEIRQSM CTGTTATCTGATACTGT\nSSLAGFIQR TGATAACATGATAGCA\nHAENKATS GCTAAAAAGAATAATG\nPSIPFLNME CATTAAGACTTATTTCT\nRKLNLSIRE GAACGTTATGAAGATG\nLSFSYSDD CTCTCACTCAGGAAAAC\nKKILNSVS AATGCTCAGAAAAAAT\nLDLFTGKM ACTGGTTACTCAGTTCT\nYSLTGPSG AAAGTTCTTTTATTGAA\nSGKSTLVK CTCTTTACTTGCTGTAA\nIISGYYKN TATTATTTGGTTCTGTA\nYFGDIYLN TTCATATATAATATTTT\nDISLRNISD AGGTGTGCTGAATGGT\nEDLNDAIY GTAGTTAGTATCGGCCA\nYLTQDDYI CTTCATTATGATTACAT\nFMDTLRFN CATATATCATTCTTCTT\nLRLANYDA TCAACGCCAGTGGAAA\nSENEIFKVL ATATAGGGGCATTGCT\nKLANLSVV AAGTGAGATCAGGCAG\nNNEPVSLD TCAATGTCTAGCCTGGC\nTHLINRGN AGGTTTTATTCAACGTC\nNYSGGQK ATGCCGAGAATAAAGC\nQRISLARLF CACATCTCCTTCAA\nLRKPAIIIID\nEATSALDY\nINESEILSSI\nRTHFPDALI\nINISHRINL\nLECSDCVY\nVLNEGNIV\nASGHFRDL\nMVSNEYIS\nGLASVTE\n540 Microcin MTLLSFGF Klebsiella 541 ATGACATTACTTTCATT\nE492 SPVFFSVM pneumoniae TGGATTTTCTCCTGTTT\nimmunity AFCIISRSK TCTTTTCAGTCATGGCG\nmodulator FYPQRTRN TTCTGTATCATTTCACG\nKVIVLILLT TAGTAAATTCTATCCGC\nFFICFLYPL AGAGAACGCGAAACAA\nTKVYLVGS AGTTATTGTTCTGATTT\nYGIFDKFY TACTAACTTTTTTTATT\nLFCFISTLI TGTTTTTTATATCCATT\nAIAINVVIL AACAAAAGTGTATCTG\nTINGAKNE GTGGGAAGTTACGGTA\nRN TATTTGACAAATTCTAC\nCTCTTTTGCTTTATTTC\nTACGTTAATTGCAATAG\nCAATTAACGTAGTGATA\nCTTACAATAAATGGAG\nCTAAGAATGAGAGAAA\nTTAG\n\nPoison-Antidote Systems”

It is possible to keep a specific microbial cells within a desired environment. This can include killing or stopping the growth of the microbial cells if they are not in the environment. These poison-antidote system, which are different from bacteriocins can be used to achieve such containment or for selective growth of microbial cell. U.S. Pat. describes several examples of poison antidote system. Nos. Nos. A poison-antidote combination may include a cytotoxic (poison), polypeptide and an antitoxin polypeptide (antidote), in one cell. A?poison nucleotide is the one used herein. A polynucleotide that encodes a poison polypeptide and an antidote Polynucleotide is a code for a poisonous polypeptide. Refers to a polynucleotide that encodes an antidote peptide.

“In some embodiments the poison polypeptide can be expressed constitutively while the antidote peptide can only be expressed under the desired conditions. Some embodiments only allow the poison polypeptide to be expressed in unfavorable conditions. The antidote polypeptide can only be expressed in desirable conditions. In some embodiments, the poison/antidote system can be configured to ensure that the microbial cells survive in desired environments but die under unfavorable conditions. In some embodiments, the poison antidote system can be configured to kill the microbial cells if they escape from an industrial environment. A poison antidote is also configured to ensure that the microbial cells survive when a vector (e.g. A plasmid encoding an antidote peptide dies when it is missing. Some embodiments encode the poison polypeptide in the host genome. The antidote peptide is encoded on a vector (such a plasmid, extrachromosomal array, episome, or minichromosome) and is only expressed when that vector is present in host cells. Some embodiments encode the poison polypeptide by a poison mononucleotide in a first vector. The antidote peptide is encoded on an antidote nucleotide in a second vector. As such, it is only expressed when that vector is present. In some embodiments, the presence or absence a recombination event (e.g. the integration of a polynucleotide sequencing encoding antidotepolynucleotide into a host genome) can determine whether the antidote oligotide is present. In some embodiments, expression of the antidote peptide is dependent on the presence of a vector or other recombination events. The poison and antidote peptide may be expressed constitutively. In some embodiments, where expression of antidotepolypeptide depends upon the presence of a vector, recombination events, or both, the expression of the poison and/or antidote can be conditional. This means that the poison polypeptide or antidote may only be expressed under conditions where the microbial cells are not desired and the antidote peptide only in those conditions.

“Exemplary microbial toxin polypeptide/antitoxin polypeptide pairs (also referred to as ?poison/antidote? Pairs that can be used in poison antidote system in conjunction with certain embodiments of this invention include, but not limited to, RelE/RelB and CcdB/CcdA. Many poison polypeptides such as RelE are extremely conserved in Gram-positive and Gram?negative bacteria and Archae. As such, they can be cytotoxic to a wide range of genetically modified and synthetic microbial cells. It is possible that an antidote peptide can inhibit the activity its poison partner in a wide range of host environments. This means that poison/antidote pairings such as those discussed herein can be used in a wide variety of genetically modified, naturally occurring and fully synthetic microbial cell types.

“A poison-antidote is different from a Bacteriocin system in that it provides an endogenous mechanism by which a microbe can kill or arrest itself. A bacteriocin provides an exogenous mechanism by which a microbe can kill or arrest another cell. A poison-antidote can only be used to kill or imprison the specific cell that produced the poison. However, some embodiments allow for a combination of a bacteriocin and poison-antidote systems. In some cases, a bacteriocin system described herein can be used to kill/arrest the growth of other cells in a culture. The poison-antidote may be used in conjunction with the bacteriocin-producing cell to stop it from growing in an unfriendly environment. The poison-antidote method can also be used to select bacteriocin-producing cells that have been genetically engineered so that they express an industrially useful molecule (an “industrially useful molecular”). In some cases, an antidote may be linked to the expression of an industrially-use molecule or bacteriocin. This can be done by either placing polynucleotides that encode the bacteriocin or the antidote or both under the control of one promoter. In some embodiments, the poison antidote system is also included in a bacteriocin-encoding microbial cell. The bacteriocin system can be used to regulate growth of microbial cells or other microbial cells in a specific environment. The poison-antidote is used for controlling microbial cells within that environment.

“Promoters”

“Promoters are well-known in the art. One or more genes can be driven by a promoter. A promoter can be used to drive the expression of a polynucleotide that encodes a desired gene product in some embodiments. A promoter can be used to drive expression of the bacteriocin polynucleotide in some embodiments. A promoter can drive expression of an immune modulator polynucleotide in some embodiments. A promoter can drive expression of a bacteriocin and an immunity modulator polynucleotide in some embodiments. A promoter can drive expression of a polynucleotide that encodes at least one of the following: a bacteriocin nucleotide, an immunity modulator, poison molecule or antidote molecular. Some promoters are able to drive transcription at all times (called “constitutive promotors?”). Some promoters are able to drive transcription in certain circumstances (conditional promoters). This could be due to the presence or absence a chemical compound, environmental condition, gene product, or stage of the cell’s cycle or other factors.

“The skilled artisan will know that depending on the expression activity desired, a promoter can be chosen and placed in cis along with the sequence to be expressed. Table 3.1-3.11 shows examples of promoters that have exemplary activities. A skilled artisan will recognize that not all promoters work with every transcriptional machine (e.g. RNA polymerases, general transcript factors, and other such things. As such, compatible species may not exist. While some promoters are described herein as compatible, it is possible that these promoters may also be used in other microorganisms.

The Biobricks foundation makes the promoters of Tables 3.1-3.11 publicly available. These promoters can be used in accordance to BioBrick, according to the Biobricks foundation Public Agreement (BPA), is encouraged.

“It is important to note that any of these?coding? “It should be appreciated that any of the?codings described herein (for instance, a bacteriocin, immunity, poison, antidote, antidote, product polynucleotide or bacteriocin) can generally be expressed under control by a desired promoter. One?coding may be used in some embodiments. A single promoter controls polynucleotide. In some cases, there may be more than one?coding. One promoter controls all polynucleotides. This could be two, three or four polynucleotides, five, six and seven polynucleotides, eight polynucleotides, nine polynucleotides, ten polynucleotides, or both. In some cases, this can be referred to as a “cocktail”. A single microorganism can produce a variety of bacteriocins. A promoter may control a bacteriocin-polynucleotide. A promoter may also control an immunity modulator in some embodiments. A promoter may control a polynucleotide that encodes a desired gene product in some embodiments. The same promoter may control both the bacteriocin and polynucleotides encoding the desired gene products in some embodiments. Different promoters may control the bacteriocin and polynucleotides encoding desired gene products in some embodiments. In some embodiments the promoter can control both the immunity modulator polynucleotide or the polynucleotide that encodes a desired gene product. In some cases, the immunity modulator and bacteriocin polynucleotide can be controlled by different promoters.

“Generally speaking, translation initiation of a transcript is controlled by sequences at or 5?” Which sequences are at or 5? End of the coding sequence for a transcript. A coding sequence may begin with a start codon that is designed to pair with an initiator transcript. Although Met (AUG is the most common start codon in naturally occurring translation systems), it will be obvious that an initiator transcript can be engineered so that it binds to any desired triplet. In certain instances, other triplets than AUG may also be used as start codons. Sequences located near the start codon may facilitate ribosomal assembly. For example, a Kozak sequence, (gcc)gccRccAUGG; SEQ ID NO. 542, where R is?A? Or?G? or?G?) A transcript may also include a?coding? in certain embodiments. A polynucleotide sequence such as a bacteriocin or immunity modulator sequence or polynucleotide that encodes a desired industrial product, includes a suitable start codon and translational sequence. Some embodiments may include multiple?coding. Each polynucleotide sequencing is placed in cis of a transcript. It contains a start codon and a translational initiation sequence. Some embodiments allow for multiple?coding. If two or more?coding sequences are placed in cis on a transcript then the two sequences are controlled by a single translation initiator sequence. They either provide a single protein that can function with both encoded cis polypeptides or provide a way to separate two cis polypeptides, such as a 2A sequence or similar. A translational intiator (tRNA) may be regulable, so that it regulates the initiation of translation for a bacteriocin or immunity modulator, poison, antidote, industrially useful molecule, and other bacteriocins.

“TABLE 3.1\nExemplary Metal-Sensitive Promoters\nSEQ\nID\nNO: Name Description Sequence\n544 BBa_I721001 Lead Promoter gaaaaccttgtcaatgaagagcgatctatg\n545 BBa_I731004 FecA promoter ttctcgttcgactcatagctgaacacaaca\n546 BBa_I760005 Cu-sensitive promoter atgacaaaattgtcat\n547 BBa_I765000 Fe promoter accaatgctgggaacggccagggcacctaa\n548 BBa_I765007 Fe and UV promoters ctgaaagcgcataccgctatggagggggtt\n549 BBa_J3902 PrFe (PI +?PII rus operon) tagatatgcctgaaagcgcataccgctatg”

“TABLE 3.2\nExemplary Cell Signaling-Responsive Promoters\nSEQ\nID\nNO: Name Description Sequence\n550 BBa_I1051 Lux cassette right promoter tgttatagtcgaatacctctggcggtgata\n551 BBa_I14015 P(Las) TetO ttttggtacactccctatcagtgatagaga\n552 BBa_I14016 P(Las) CIO ctttttggtacactacctctggcggtgata\n553 BBa_I14017 P(Rhl) tacgcaagaaaatggtttgttatagtcgaa\n554 BBa_I739105 Double Promoter (LuxR/HSL, cgtgcgtgttgataacaccgtgcgtgttga\npositive/cI, negative)\n555 BBa_I746104 P2 promoter in agr operon agattgtactaaatcgtataatgacagtga\nfrom S. aureus\n556 BBa_I751501 plux-cI hybrid promoter gtgttgatgcttttatcaccgccagtggta\n557 BBa_I751502 plux-lac hybrid promoter agtgtgtggaattgtgagcggataacaatt\n558 BBa_I761011 CinR, CinL and glucose acatcttaaaagttttagtatcatattcgt\ncontrolled promotor\n559 BBa_J06403 RhIR promoter repressible by tacgcaagaaaatggtttgttatagtcgaa\nCI\n560 BBa_J102001 Reverse Lux Promoter tcttgcgtaaacctgtacgatcctacaggt\n561 BBa_J64000 rhlI promoter atcctcctttagtcttccccctcatgtgtg\n562 BBa_J64010 lasI promoter taaaattatgaaatttgcataaattcttca\n563 BBa_J64067 LuxR +?3OC6HSL independent gtgttgactattttacctctggcggtgata\nR0065\n564 BBa_J64712 LasR/LasI Inducible & gaaatctggcagtttttggtacacgaaagc\nRHLR/RHLI repressible\nPromoter\n565 BBa_K091107 pLux/cI Hybrid Promoter acaccgtgcgtgttgatatagtcgaataaa\n566 BBa_K091117 pLas promoter aaaattatgaaatttgtataaattcttcag\n567 BBa_K091143 pLas/cI Hybrid Promoter ggttctttttggtacctctggcggtgataa\n568 BBa_K091146 pLas/Lux Hybrid Promoter tgtaggatcgtacaggtataaattcttcag\n569 BBa_K091156 pLux caagaaaatggtttgttatagtcgaataaa\n570 BBa_K091157 pLux/Las Hybrid Promoter ctatctcatttgctagtatagtcgaataaa\n571 BBa_K145150 Hybrid promoter: HSL-LuxR tagtttataatttaagtgttctttaatttc\nactivated, P22 C2 repressed\n572 BBa_K266000 PAI +?LasR ->?LuxI (AI) caccttcgggtgggcctttctgcgtttata\n573 BBa_K266005 PAI +?LasR ->?LasI & AI + aataactctgatagtgctagtgtagatctc\nLuxR –|LasI\n574 BBa_K266006 PAI +?LasR ->?LasI +?GFP & caccttcgggtgggcctttctgcgtttata\nAI +?LuxR –|LasI +?GFP\n575 BBa_K266007 Complex QS ->?LuxI & LasI caccttcgggtgggcctttctgcgtttata\ncircuit\n576 BBa_K658006 position 3 mutated promoter caagaaaatggtttgttatagtcgaataaa\nlux pR-3 (luxR & HSL\nregulated)\n577 BBa_K658007 position 5 mutated promoter caagaaaatggtttgttatagtcgaataaa\nlux pR-5 (luxR & HSL\nregulated)\n578 BBa_K658008 position 3&5 mutated caagaaaatggtttgttatagtcgaataaa\npromoter lux pR-3/5 (luxR &\nHSL regulated)\n579 BBa_R0061 Promoter (HSL-mediated luxR ttgacacctgtaggatcgtacaggtataat\nrepressor)\n580 BBa_R0062 Promoter (luxR & HSL caagaaaatggtttgttatagtcgaataaa\nregulated — lux pR)\n581 BBa_R0063 Promoter (luxR & HSL cacgcaaaacttgcgacaaacaataggtaa\nregulated – lux pL)\n582 BBa_R0071 Promoter (Rh1R & C4-HSL gttagctttcgaattggctaaaaagtgttc\nregulated)\n583 BBa_R0078 Promoter (cinR and HSL ccattctgctttccacgaacttgaaaacgc\nregulated)\n584 BBa_R0079 Promoter (LasR & PAI ggccgcgggttctttttggtacacgaaagc\nregulated)\n585 BBa_R1062 Promoter, Standard (luxR and aagaaaatggtttgttgatactcgaataaa\nHSL regulated — lux pR)”

“TABLE 3.3\nExemplary Constitutive E. coli ?70 Promoters\nSEQ\nID\nNO: Name Description Sequence\n586 BBa_I14018 P(Bla) gtttatacataggcgagtactctgttatgg\n587 BBa_I14033 P(Cat) agaggttccaactttcaccataatgaaaca\n588 BBa_I14034 P(Kat) taaacaactaacggacaattctacctaaca\n589 BBa_I732021 Template for Building Primer acatcaagccaaattaaacaggattaacac\nFamily Member\n590 BBa_I742126 Reverse lambda cI-regulated gaggtaaaatagtcaacacgcacggtgtta\npromoter\n591 BBa_J01006 Key Promoter absorbs 3 caggccggaataactccctataatgcgcca\n592 BBa_J23100 constitutive promoter family ggctagctcagtcctaggtacagtgctagc\nmember\n593 BBa_J23101 constitutive promoter family agctagctcagtcctaggtattatgctagc\nmember\n594 BBa_J23102 constitutive promoter family agctagctcagtcctaggtactgtgctagc\nmember\n595 BBa_J23103 constitutive promoter family agctagctcagtcctagggattatgctagc\nmember\n596 BBa_J23104 constitutive promoter family agctagctcagtcctaggtattgtgctagc\nmember\n597 BBa_J23105 constitutive promoter family ggctagctcagtcctaggtactatgctagc\nmember\n598 BBa_J23106 constitutive promoter family ggctagctcagtcctaggtatagtgctagc\nmember\n599 BBa_J23107 constitutive promoter family ggctagctcagccctaggtattatgctagc\nmember\n600 BBa_J23108 constitutive promoter family agctagctcagtcctaggtataatgctagc\nmember\n601 BBa_J23109 constitutive promoter family agctagctcagtcctagggactgtgctagc\nmember\n602 BBa_J23110 constitutive promoter family ggctagctcagtcctaggtacaatgctagc\nmember\n603 BBa_J23111 constitutive promoter family ggctagctcagtcctaggtatagtgctagc\nmember\n604 BBa_J23112 constitutive promoter family agctagctcagtcctagggattatgctagc\nmember\n605 BBa_J23113 constitutive promoter family ggctagctcagtcctagggattatgctagc\nmember\n606 BBa_J23114 constitutive promoter family ggctagctcagtcctaggtacaatgctagc\nmember\n607 BBa_J23115 constitutive promoter family agctagctcagcccttggtacaatgctagc\nmember\n608 BBa_J23116 constitutive promoter family agctagctcagtcctagggactatgctagc\nmember\n609 BBa_J23117 constitutive promoter family agctagctcagtcctagggattgtgctagc\nmember\n610 BBa_J23118 constitutive promoter family ggctagctcagtcctaggtattgtgctagc\nmember\n611 BBa_J23119 constitutive promoter family agctagctcagtcctaggtataatgctagc\nmember\n612 BBa_J23150 1bp mutant from J23107 ggctagctcagtcctaggtattatgctagc\n613 BBa_J23151 1bp mutant from J23114 ggctagctcagtcctaggtacaatgctagc\n614 BBa_J44002 pBAD reverse aaagtgtgacgccgtgcaaataatcaatgt\n615 BBa_J48104 NikR promoter, a protein of gacgaatacttaaaatcgtcatacttattt\nthe ribbon helix-helix family of\ntrancription factors that repress\nexpre\n616 BBa_J54200 lacq_Promoter aaacctttcgcggtatggcatgatagcgcc\n617 BBa_J56015 lacIQ – promoter sequence tgatagcgcccggaagagagtcaattcagg\n618 BBa_J64951 E. Coli CreABCD phosphate ttatttaccgtgacgaactaattgctcgtg\nsensing operon promoter\n619 BBa_K088007 GlnRS promoter catacgccgttatacgttgtttacgctttg\n620 BBa_K119000 Constitutive weak promoter of ttatgcttccggctcgtatgttgtgtggac\nlacZ\n621 BBa_K119001 Mutated LacZ promoter ttatgcttccggctcgtatggtgtgtggac\n622 BBa_K137029 constitutive promoter with atatatatatatatataatggaagcgtttt\n(TA)10 between ?10 and ?35\nelements\n623 BBa_K137030 constitutive promoter with atatatatatatatataatggaagcgtttt\n(TA)9 between ?10 and ?35\nelements\n624 BBa_K137031 constitutive promoter with ccccgaaagcttaagaatataattgtaagc\n(C)10 between ?10 and ?35\nelements\n625 BBa_K137032 constitutive promoter with ccccgaaagcttaagaatataattgtaagc\n(C)12 between ?10 and ?35\nelements\n626 BBa_K137085 optimized (TA) repeat tgacaatatatatatatatataatgctagc\nconstitutive promoter with 13\nbp between ?10 and ?35\nelements\n627 BBa_K137086 optimized (TA) repeat acaatatatatatatatatataatgctagc\nconstitutive promoter with 15\nbp between ?10 and ?35\nelements\n628 BBa_K137087 optimized (TA) repeat aatatatatatatatatatataatgctagc\nconstitutive promoter with 17\nbp between ?10 and ?35\nelements\n629 BBa_K137088 optimized (TA) repeat tatatatatatatatatatataatgctagc\nconstitutive promoter with 19\nbp between ?10 and ?35\nelements\n630 BBa_K137089 optimized (TA) repeat tatatatatatatatatatataatgctagc\nconstitutive promoter with 21\nbp between ?10 and ?35\nelements\n631 BBa_K137090 optimized (A) repeat aaaaaaaaaaaaaaaaaatataatgctagc\nconstitutive promoter with 17\nbp between ?10 and ?35\nelements\n632 BBa_K137091 optimized (A) repeat aaaaaaaaaaaaaaaaaatataatgctagc\nconstitutive promoter with 18\nbp between ?10 and ?35\nelements\n633 BBa_K256002 J23101:GFP caccttcgggtgggcctttctgcgtttata\n634 BBa_K256018 J23119:IFP caccttcgggtgggcctttctgcgtttata\n635 BBa_K256020 J23119:HO1 caccttcgggtgggcctttctgcgtttata\n636 BBa_K256033 Infrared signal reporter caccttcgggtgggcctttctgcgtttata\n(J23119:IFP:J23119:HO1)\n637 BBa_K292000 Double terminator + ggctagctcagtcctaggtacagtgctagc\nconstitutive promoter\n638 BBa_K292001 Double terminator + tgctagctactagagattaaagaggagaaa\nConstitutive promoter +?Strong\nRBS\n639 BBa_K418000 IPTG inducible Lac promoter ttgtgagcggataacaagatactgagcaca\ncassette\n640 BBa_K418002 IPTG inducible Lac promoter ttgtgagcggataacaagatactgagcaca\ncassette\n641 BBa_K418003 IPTG inducible Lac promoter ttgtgagcggataacaagatactgagcaca\ncassette\n642 BBa_M13101 M13K07 gene I promoter cctgtttttatgttattctctctgtaaagg\n643 BBa_M13102 M13K07 gene II promoter aaatatttgcttatacaatcttcctgtttt\n644 BBa_M13103 M13K07 gene III promoter gctgataaaccgatacaattaaaggctcct\n645 BBa_M13104 M13K07 gene IV promoter ctcttctcagcgtcttaatctaagctatcg\n646 BBa_M13105 M13K07 gene V promoter atgagccagttcttaaaatcgcataaggta\n647 BBa_M13106 M13K07 gene VI promoter ctattgattgtgacaaaataaacttattcc\n648 BBa_M13108 M13K07 gene VIII promoter gtttcgcgcttggtataatcgctgggggtc\n649 BBa_M13110 M13110 ctttgcttctgactataatagtcagggtaa\n650 BBa_M31519 Modified promoter sequence of aaaccgatacaattaaaggctcctgctagc\ng3.\n651 BBa_R1074 Constitutive Promoter I caccacactgatagtgctagtgtagatcac\n652 BBa_R1075 Constitutive Promoter II gccggaataactccctataatgcgccacca\n653 BBa_S03331 –Specify Parts List– ttgacaagcttttcctcagctccgtaaact”

“TABLE 3.4\nExemplary Constitutive E. coli ?s Promoters\nSEQ\nID\nNO: Name Description Sequence\n654 BBa_J45992 Full-length stationary phase ggtttcaaaattgtgatctatatttaacaa\nosmY promoter\n655 BBa_J45993 Minimal stationary phase osmY ggtttcaaaattgtgatctatatttaacaa\npromoter”

“TABLE 3.5\nExemplary Constitutive E. coli ?32 Promoters\nSEQ\nID\nNO: Name Description Sequence\n656 BBa_J45504 htpG Heat Shock Promoter tctattccaataaagaaatcttcctgcgtg”

“TABLE 3.6\nExemplary Constitutive B. subtilis ?A Promoters\nSEQ\nID\nNO: Name Description Sequence\n657 BBa_K143012 Promoter veg a constitutive aaaaatgggctcgtgttgtacaataaatgt\npromoter for B. subtilis\n658 BBa_K143013 Promoter 43 a constitutive aaaaaaagcgcgcgattatgtaaaatataa\npromoter for B. subtilis\n659 BBa_K780003 Strong constitutive promoter aattgcagtaggcatgacaaaatggactca\nfor Bacillus subtilis\n660 BBa_K823000 PliaG caagcttttcctttataatagaatgaatga\n661 BBa_K823002 PlepA tctaagctagtgtattttgcgtttaatagt\n662 BBa_K823003 Pveg aatgggctcgtgttgtacaataaatgtagt”

“TABLE 3.7\nExemplary Constitutive B. subtilis ?B Promoters\nSEQ\nID\nNO: Name Description Sequence\n663 BBa_K143010 Promoter ctc for B. subtilis atccttatcgttatgggtattgtttgtaat\n664 BBa_K143011 Promoter gsiB for B. subtilis taaaagaattgtgagcgggaatacaacaac\n665 BBa_K143013 Promoter 43 a constitutive aaaaaaagcgcgcgattatgtaaaatataa\npromoter for B. subtilis”

“TABLE 3.8\nExemplary Constitutive Promoters from miscellaneous prokaryotes\nSEQ\nID\nNO: Name Description Sequence\n666 a_K112706 Pspv2 from Salmonella tacaaaataattcccctgcaaacattatca\n667 BBa_K112707 Pspv from Salmonella tacaaaataattcccctgcaaacattatcg”

“TABLE 3.9\nExemplary Constitutive Promoters from bacteriophage T7\nSEQ\nID\nNO: Name Description Sequence\n668 BBa_I712074 T7 promoter (strong agggaatacaagctacttgttctttttgca\npromoter from T7\nbacteriophage)\n669 BBa_I719005 T7 Promoter taatacgactcactatagggaga\n670 BBa_J34814 T7 Promoter gaatttaatacgactcactatagggaga\n671 BBa_J64997 T7 consensus ?10 and rest taatacgactcactatagg\n672 BBa_K113010 overlapping T7 promoter gagtcgtattaatacgactcactatagggg\n673 BBa_K113011 more overlapping T7 agtgagtcgtactacgactcactatagggg\npromoter\n674 BBa_K113012 weaken overlapping T7 gagtcgtattaatacgactctctatagggg\npromoter\n675 BBa_R0085 T7 Consensus Promoter taatacgactcactatagggaga\nSequence\n676 BBa_R0180 T7 RNAP promoter ttatacgactcactatagggaga\n677 BBa_R0181 T7 RNAP promoter gaatacgactcactatagggaga\n678 BBa_R0182 T7 RNAP promoter taatacgtctcactatagggaga\n679 BBa_R0183 T7 RNAP promoter tcatacgactcactatagggaga\n680 BBa_Z0251 T7 strong promoter taatacgactcactatagggagaccacaac\n681 BBa_Z0252 T7 weak binding and taattgaactcactaaagggagaccacagc\nprocessivity\n682 BBa_Z0253 T7 weak binding promoter cgaagtaatacgactcactattagggaaga”

“TABLE 3.10\nExemplary Constitutive Promoters from yeast\nSEQ\nID\nNO: Name Description Sequence\n683 BBa_I766555 pCyc (Medium) Promoter acaaacacaaatacacacactaaattaata\n684 BBa_I766556 pAdh (Strong) Promoter ccaagcatacaatcaactatctcatataca\n685 BBa_I766557 pSte5 (Weak) Promoter gatacaggatacagcggaaacaacttttaa\n686 BBa_J63005 yeast ADH1 promoter tttcaagctataccaagcatacaatcaact\n687 BBa_K105027 cyc100 minimal promoter cctttgcagcataaattactatacttctat\n688 BBa_K105028 cyc70 minimal promoter cctttgcagcataaattactatacttctat\n689 BBa_K105029 cyc43 minimal promoter cctttgcagcataaattactatacttctat\n690 BBa_K105030 cyc28 minimal promoter cctttgcagcataaattactatacttctat\n691 BBa_K105031 cyc16 minimal promoter cctttgcagcataaattactatacttctat\n692 BBa_K122000 pPGK1 ttatctactttttacaacaaatataaaaca\n693 BBa_K124000 pCYC Yeast Promoter acaaacacaaatacacacactaaattaata\n694 BBa_K124002 Yeast GPD (TDH3) gtttcgaataaacacacataaacaaacaaa\nPromoter\n695 BBa_K319005 yeast mid-length ADH1 ccaagcatacaatcaactatctcatataca\npromoter\n696 BBa_M31201 Yeast CLB1 promoter accatcaaaggaagctttaatcttctcata\nregion, G2/M cell cycle\nspecific”

“TABLE 3.11\nExemplary Constitutive Promoters from miscellaneous\neukaryotes\nSEQ\nID\nNO: Name Description Sequence\n697 BBa_I712004 CMV promoter agaacccactgcttactggcttatcgaaat\n698 BBa_K076017 Ubc Promoter ggccgtttttggcttttttgttagacgaag”

The above-referenced promotors are only an example. A skilled artisan will quickly recognize that there are many variations of the promoters mentioned above, as well as many other promoters (including those isolated from naturally occurring organisms, variations thereof, or fully synthetic promoters), which can be easily used in accordance to some embodiments.

“Regulation of Gene Activity.”

“Gene activity can either be increased or decreased by regulating it. One embodiment of the gene product whose activity is controlled includes a bacteriocin or immunity modulator, poison molecule, antidote, molecule, and industrially useful molecules. One gene regulation system may regulate multiple gene products. Some embodiments regulate gene activity at the level gene expression. Some embodiments regulate gene activity at the transcriptional level. This could be done by activating or suppressing a promoter. Some embodiments regulate gene activity at the post-transcriptional levels, such as regulation of RNA stability. Some embodiments regulate gene activity at the translational level. This could be done by regulating the initiation of translation. Some embodiments regulate gene activity at the post-translational levels, such as regulation of polypeptide stability or post-translational modifications of the polypeptide or binding an inhibitor to that polypeptide.

“In some embodiments gene activity is increased. Some embodiments increase the activity of at least one of the following: bacteriocins, immunity modulators, industrially useful molecules, poison molecule or antidote molecules. The idea behind increasing gene activity is that it can be directly activated or decreased by an inhibitor of gene activation. Some embodiments activate gene activity by at least one of the following: Inducing promoter activation, inhibiting transcriptional repressors, increasing RNA stability or inducing a pre-transcriptional inhibitor (for instance, inducing a ribozyme (or antisense) oligonucleotide), initiating translation (for instance, via a regulatable transcriptomic (tRNA), or inducing a desired posttranslational modification or inhibiting an inhibitor (for ase that is directed to a protein coding gene) A compound that is present in the desired environment can induce a promoter. An iron-sensitive promoter can be used to induce transcription, as an example. A compound found in the desired culture medium may inhibit a transcriptional regulator. Tetracycline, for example, can be found in an environment to inhibit the transcriptional repressor and allow activity from the promoter tetO. A compound that is only found outside of the desired culture medium can induce transcription in some embodiments.

“In some embodiments, gene activation is decreased. The concept of decreasing gene activity is possible by either directly inducing gene activity or by decreasing activity of an activator. Some embodiments reduce gene activity, while maintaining some activity. Some embodiments completely inhibit gene activity. Some embodiments decrease gene activity by at least one of the following: activating a transcriptional regulator, inhibiting promoter activation, decreasing RNA stability or activating post-transcriptional inhibitors (e.g., expressing an antisense or ribozyme oligonucleotide), inactivating polypeptides (e.g., binding an inhibitor, or using a polypeptide specific protease), and failing to correctly localize polypeptides (e. Failure to secrete bacteriocin. Some embodiments reduce gene activity by removing a particular gene from a desired place, such as by using a FLP/FRT or crelox cassette to excise a gene or by the loss or degradation a plasmid. A gene product (e.g. A polypeptide or a product made by a gene products (e.g. The product of an enzyme reaction that inhibits further gene activity (e.g. a negative feedback loop).”

“Genetic Modifications of Microorganisms”

“Techniques for genetically altering microorganisms is well-known in the art. A microorganism can be genetically modified to contain a nucleic acid sequence that regulates the expression and encoding of at least one of the following: bacteriocins; immunity modulators; industrially useful molecules; poison molecules or antidote chemicals. Microorganisms can receive polynucleotides. They can either be stably integrated into their chromosomes or exist without the genome, such as in a plasmid or extrachromosomal array.

“Exemplary vectors to genetically modify microbial cells include viruses, plasmids and transposable element. It will also be apparent that complete microbial genomes containing desired sequences can be synthesized in a cell (see, for example, Gibson et al. Gibson et al. Science 329, 52-56 (2010). In some embodiments, a portion of a microbial gene (or a part thereof) is synthesized and then introduced into a microbial cells.

It is possible to genetically modify a microorganism to produce a desired type or spectrum of immunity modulator activity or bacteriocin. A cassette is available for inserting desired bacteriocin or immunity modulator polynucleotides in a polynucleotide sequencing. Examples of cassettes include, among others, a Cre/lox cassette and FLP/FRT cassette. Some embodiments of the cassette are placed on a DNA plasmid so that the desired combination of bacteriocin/or immunity modulator can be easily introduced to the microbial cells. Some embodiments position the cassette in the genome of a microbial cell so that the cassette with the desired combination of bacteriocin/or immunity modulator can be introduced to the desired place.

“Culture Media”

“Microbial culture environments may include a variety of media such as feedstocks. The application will determine the choice of the right culture medium. The conditions of a culture medium can include chemical composition as well as temperature, light levels, pH, CO2 and other factors.

“In some embodiments, a genetically engineered organism is added to a culture medium that contains other microorganisms as well as at least one feedstock. Some embodiments contain a compound that stimulates the activity of a bacteriocin or immunity modulator. In some cases, the culture medium contains a compound that inhibits or suppresses the expression or activity of a bacteriocin or immunity modulator. A compound that stimulates the activity is found outside the feedstock in some embodiments. A compound that inhibits the activity the immunity modulator may be present in some embodiments but not in the feedstock.

“The term “feedstock” is used in a broad sense to mean material that can be consumed, fermented, purified or modified by microbial organisms. “Feedstock” is used in this context in a broad meaning to include material that can be consumed or fermented, purified and modified by microbial organisms. As such, ?feedstock? It does not include food or other food products. A ‘feedstock’ is a term that can be used to refer to food products. is a type of culture medium. As such, ‘culture medium’ is used herein. It includes, but is not limited to, feedstock. As such, feedstocks are also considered when a?culture medium is mentioned. Feedstocks are also explicitly contemplated when a?culture medium” is mentioned.

“Genetically Engineered Microbial Cells.”

“In some instances, genetically modified cells for microbial growth are available. You can configure genetically modified microbial cell for many purposes. Some embodiments of microbial cells include genetic modifications that regulate at least one of the following: immunity modulators, bacteriocins or mollycose, poison molecules, antidote chemicals, and bacteriocins. Some embodiments include genetic modifications in microbial cells to regulate the expression bacteriocins. Some embodiments include genetic modifications in microbial cells to regulate expression of immunity modulators.

“In some embodiments, genetically modified microbial cell are modified to make a product. The product may be a gene product in some embodiments. For example, a polypeptide, or RNA. Polynucleotide?coding is a result. Sequence can be used to refer to sequences that encode either a polypeptide, or an RNA. Microbial cells can be programmed to produce one or more genes that help to synthesize a desired product. These include a carbohydrate or biofuel, lipid or small molecule. The activity of one or more genes from the microbial cells can be used to synthesize the product in some embodiments. Optionally, the product may also be synthesized by the activity of one or several gene products from one or more other microorganisms. Microbial cells can be programmed to remove or decontaminate one or more substances from a media, such as a feedstock, in some embodiments. One or more gene products from microbial cells can help to decontaminate the environment. Microbial cells can be programmed to search for materials, such as iron and rare earth metals.

“Controlling Microbial Cell Growth”

“In some instances, genetically modified microbial cell lines are used to regulate growth of other microbial species. In some cases, the microbial cell regulates the growth of other microbial cell strains or species, such as their own clones. In some instances, the microbial cell regulates the growth of microbial strains or species, such as invaders. A bacteriocin is secreted by microbial cells to regulate the growth of other microbial plants. Each microbial cell’s regulation can be affected by its expression (or absence thereof) of an immune modulator that has protective effects against particular bacteriocin.

“As used herein, a desired cell? The like and microbial cells with at least one characteristic are microbial cells that have at least one characteristic. In some embodiments, a desired cell is in an appropriate environment, for example its industrially-applicable feedstock. A desired cell can be a cell that has been positively selected for. This could include a cell with high levels of useful gene products or that has undergone particular recombination. A desired cell can be a cell that is capable of neutralizing contaminating cells such as pathogenic cells. A desired cell can be positively selected by the expression of an immunity modulator that corresponds to at least one bacteriocin. It is possible that a microbial cell which can neutralize other microbial cells without a similar neutralizing function would have a competitive advantage, but this theory is not binding. In some embodiments, the ability to neutralize other cells is what makes a desired cell desirable. A bacteriocin or a related immunity modulator can be used to positively select a desired cell.

“As used herein ?undesired cell? The microbial cells with at least one characteristic that makes survival, growth, and proliferation unfavorable are called the undesired cell. The undesired cell may be a invading microbial cells, such as a contaminating or invasive cell that has entered a culture medium. In some embodiments, an undesired cell has escaped from an appropriate culture medium, for example its industrially-applicable feedstock. An undesired cell may have lost a specific plasmid or failed to undergo a particular event of recombination. An undesired cell may not produce or produce the desired gene product in some embodiments. An undesired cell may be selected against in some embodiments. An undesired cell can be selected against by reducing its expression or activity of an immunity modifier that protects against abacin in the environment. An undesired cell can be selected against by reducing its expression or activity of an immune modulator that protects against the secretion of bacteriocin by the cell and its clones. An undesired cell can be selected against in some embodiments by decreasing the expression of bacteriocin in the cell, which puts it at a competitive disadvantage over other microbial cell types.

“FIG. “FIG. A first microbial cells is sometimes provided in some embodiments. Some embodiments provide a first microbial cells that secrete an active bacteriocin 100. Some embodiments do not want the first microbial cells 102. In some embodiments, for example, one or more of the earliest microbial cells being outside their industrial environment or lacking a desired environment condition for the first microbe can render the first microbe undesirable in a specific environment at a particular moment. If the first microbial cells are not desired, the immunity modulator, which corresponds to the bacteriocin, can be inactive. One or more of the immunity modulator promoters can be inactive, and an immunity modulator transcriptional regulator can be active. Post-transcriptional silencing can also occur (e.g. A ribozyme, antisense, or regulatable tRNA may occur. Post-transcriptional silencing (e.g. Site-specific protease or silencing posttranslational modification can occur, or a vector encoding a immunity modulator may be absent. If the first cell doesn’t have an active immune modulator, it is neutralized by the bacteriocin (142) produced by other cells in culture. A second microbial cell may grow 192 after the first cell has been neutralized.

“In some embodiments, it is desirable to have the first microbial cells 106. One or more of the desired microbial cells can be found within an industrial environment. The first microbial cells may also have undergone a recombination process or contain a specific vector. This makes the cell desirable in a particular environment. When a microbial cell produces an active immune modulator, it can do so when desired. In some cases, the first microbial cells can have one or more of the following: a constitutive inducer for immunity modulator polynucleotide; an activated promoter (but not necessarily constitutive); an inactive repressor for immunity modulator transcription; a regulatable transcript that is induced for production of the immune modulator; absence of post-translational or post-transcriptional silencing or a vector encoding it 136. The first microbial cells can survive in the absence of bacteriocin from the first microbial cells. A second microbial cells can either grow to 192 or be neutralized by the bacteriocin produced by the first microbial. This depends on whether or not the second microbial has 176 immunity modulator activity.

“In some embodiments, the second cell of the microbial family is desired 152. One or more desired recombination events may have occurred in the second cell. A desired vector can be present in the second cell. The second cell produces a product of greater value (e.g. A positive feedback loop, or the immune locus and desired product being under the exact same transcriptional control can make the second microbial cells desirable 162. The second microbial cells can be used to provide immunity modulator activity in order to protect against the bacteriocin or bacterocins produced by the first. In some cases, the second microbial cells can be designed so that the immunity modulator promoter (for example, a constitutive one) is active, an immunity modulator transcriptional regulator is inactive, an immunity modulator transcriptional suppressor is inactive, an immunity modulator transcriptional regulatory repressor is not inactive, a regulatable transcript (for the facilitation of the expression of an immunity modulator), and a lack post-translational silence (e.g. by site-specific protease of the immunity modulator or a vector encoding a immunity modulator may be present 182. In certain embodiments, the immunity modulator activity can be provided so that the second microbial cells can survive 192.

“In some embodiments, a secondary microbial cell may not be desired 156. One or more of the second cells could be an intruder (e.g. A contaminating cell, an unfavorable environmental condition for the second microbe (e.g. The presence or absence of an undesired condition or compound, the second microbial cells having produced product but not more (e.g. A negative feedback loop, or an immune modulator locus and desired products locus being under the exact same transcriptional control. Transcript levels are not desirable (e.g. The second microbial cells may become unattractive if they are unable to produce the desired product. In some embodiments, the immunity modulator activity of the second microbial cells can be insufficient or absent. 176 One or more of the immune modulator promoters can be inactive, and an immunity modulator transcriptional regulator can be active. Post-transcriptional silencing can also occur (e.g. A ribozyme, antisense oligonucleotide or ribozyme can be used to induce the immunity modulator. Post-transcriptional silencing can also occur (e.g. Site-specific protease or silencing posttranslational modification can occur, or an absence of a vector encoding the immunity modulator may occur 186. Some embodiments allow the first microbial cells to secrete bacteriocin activity 100. In some embodiments, the bacteriocin can kill the second microbial cells.

“One skilled in art will recognize that the steps, functions, and structures disclosed herein may be executed in a different order. The outlined functions or structures are just examples. Some functions and structure may be combined into smaller functions or structures. Other functions and/or structures can also be added without affecting the essence of the disclosed embodiments.

It can be used to control the growth of other microbial cell cultures in a culture for a wide range of genetically modified microbial organisms. A microbial cell can control the growth of other cells in the culture, according to some embodiments. Table 4 shows some examples of functions and configurations that a first microbial Cell can use to control the growth one or more other Microbial Cells according to certain embodiments.

“TABLE 4\nExemplary uses of bacteriocin systems in genetically modified\nmicrobial cells according to some embodiments herein\nExemplary Exemplary configurations\nFunction (according to some embodiments)\nBiological Immunity modulator activity\ncontainment: only in the desired culture\nmedium, but not outside and\nbacteriocin activity at least\noutside of the desired culture\nmedium; escape of the\nbacteriocin producing cell\noutside the desired culture\nenvironment results in cytotoxicity\nor growth inhibition of\nthe bacteriocin producing cell\nGenetic guard Bacteriocin constitutively produced;\ngenetic guard microbial organism does\nnot produce gene products for\nmodulating industrial process\nof interest; immunity\nmodulator constitutively produced\n(e.g under control of\nconstitutive promoter) and/or\ngenetic guard microbial\norganism is insensitive to\nthe bacteriocin (e.g. a S.\ncerevisiae genetic guard\nproducing bacteriocins that\ntarget E. coli)\nSelection of Desired recombination event\nrecombinants: causes an immunity\nmodulator to be restored in a\nbacteriocin-expressing host.\nAlternatively the immunity modulator\ncan be restored only\nafter the desired recombination event.\nVector stability: Immunity modulator (or at least\none gene essential for\nimmunity is encoded on a\nplasmid, and a corresponding\nbacteriocin locus is encoded on\nchromosome); clones that\nlose the desired plasmid lack\nimmunity and are neutralized\nby the bacteriocin\nMinimization of Immunity modulator activity\ngenetic drift dependent on production of\nindustrial product (e.g. immunity\nmodulator expression\ncontrolled by an operon, in which\na repressor is active in\nthe absence of industrial product,\nand inactive in the\npresence of industrial product);\nif a mutation causes the\nmicrobial organism’s production\nof industrial product to\nfall below a desired level or\ncease, the microbial organism\nceases to produce immunity\nmodulator, and is neutralized\nby the bacteriocin.\nSelection for Immunity modulator is\nmicrobes presenting co-expressed with the gene of\na high yield interest; microbial organisms\nof expression producing high levels of\nexpression (and/or gene product of interest can\nexpressing be selected by increasing\nclones) bacteriocin concentration;\nmicrobial organisms producing\nlow levels of gene product of\ninterest (e.g. Low industrial fitness? are neutralized\nDestruction during Desired microbial cells\nfermentation constitutively express at least one\nof contaminating type of bacteriocin; secreted\nmicrobes. bacteriocins neutralize\ninvading microbial cells\nDesired microbial cells express\nat least one type of\nbacteriocin when in the\ndesired environment (e.g.\nbacteriocin is under the control\nof an inducible promoter\nthat is activated by an\nintermediate of the fermentation\nprocess); secreted bacteriocins\nneutralize contaminating\ncells\nControl of the Immunity modulator activity\nratio of a is repressed by accumulated\nmicrobial flora, product made by a microbial\ncell; bacteriocins secreted by\nthe microbial cell (or other cells)\nneutralize the microbial\ncell”

“FIG. “FIG. 2” is a diagram showing a genetically engineered cell that controls the growth of at most one other microbial cells according to certain embodiments. A first microbial cells 200 may contain a bacteriocin and an immunity modulator polynucleotide. Optionally, the bacteriocin mononucleotide may be integrated into the cell’s DNA. The immunity modulator polynucleotide may be optionally integrated into a cell’s plasmid. Unwanted clones of the cell 210 can be created in some embodiments (a non-expressing clone). can be devoid of immunity modulator activity and can optionally lack bacteriocin activation. The non-expressing clone can be neutralized by the bacteriocin activity in the first microbial cells 200. An undesired cell clone 220 may lose a plasmid containing the immunity modulator polynucleotide. The undesired 220 clone can be neutralized by the bacteriocin activity in the first microbial cells 200. Some embodiments allow the microbial cells 230 to escape from the desired environment. This can cause the clone’s immunity modulator activity to be ineffective. The escaped cells 230 and/or the clones can neutralize the cell 230. The escaped cell 230 may also include a poison-antidote mechanism to kill the escaped cells upon their escape.

“FIG. “FIG. A first bacteriocin-polynucleotide can be found in the genetically engineered microbial cells 300. The second genetically engineered microbe 310 can contain a second bacteriocin oligotide. Each of the genetically engineered microbial cell (300 and 301) can contain a first immunity modulator and second immunity modulator polynucleotides. These polynucleotides encode resistance to the first bacteriocin. The second genetically engineered microbe 310 can become unwelcome and lose its first immunity modulator activity through any of the mechanisms described herein. It will then be controlled by the 300-generated first bacteriocin. The first bacteriocin 300 from the first genetically-engineered microbial cells and the second bacteriocin 310 from the second genetically-engineered microbial cells can neutralize an intruder cell.

“FIG. “FIG. The first genetically engineered cells 400 may contain at least one bacteriocin encoding first bacteriocin and at most one second bacteriocin encoding second bacteriocin. The first genetically engineered cells 400 can produce the first anti-invader cell bacteriocin 410. The second bacteriocin can be produced by the first genetically engineered cells 410 to neutralize a second intruder cell 420. Some embodiments allow the first invader to be of a different species or strain than the second invader. Some embodiments show that the first invader cells respond differently to different levels of bacteriocin activity from the second invader cells. Some embodiments show that the first invader cell occupies a different ecological niche to the second invader.

“FIG. “FIG.5” is a flow diagram that illustrates methods for controlling the growth at least one second microbial cells in culture, according to certain embodiments. This could include culturing a first-microbial cells in a culture medium that contains a second microbial. The conditions are such that the first microbial cells produce a bacteriocin sufficient to control growth of the second cell. Optionally, the first microbial cells can be maintained continuously for a time of 520. Some embodiments require that the first microbial cells are maintained continuously for at least three days. For example, 3, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 25, 35, 45, 55, 65, 70 or 75. 530. A change in culture media that indicates the presence or an increase in activity of a third microorganism can be detected. In response to the production of a second bacteriocin, the first microbial cells can be re-engineered. This will allow the third microbial cells to grow. In the conditions where the first microbial cells produce a bacteriocin sufficient to control growth of the second microbial cells, the re-engineered microbial cells can be grown in culture. You can continue to culture the re-engineered first microbial cell for up to 560 days. Some embodiments require that the culture of the re-engineered microorganism cell be maintained for at least three days. This could include at least 3, 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 25, 40, 45, 55, 65, 70 or 75.

“In certain embodiments, a first-microbial cell can control growth of a second microbe. A first microbial cells can control the growth of another microbial cells of the same strain. The cells of the strain may contain a bacteriocin and an immunity modulator polynucleotide. If the immunity modulator is expressed, it protects against the bacteriocin. If a strain clone loses the expression of the immunity modator, it can be neutralized with bacteriocin activity of the same strain. In some embodiments, immunity modulator polynucleotide may be in cis with the bacteriocin oligotide. This means that even if both the bacteriocin and immunity modulator polynucleotides are eliminated (e.g. If a plasmid has been lost or an FLP-FRT cassette has been excised, bacteriocin activity can still neutralize the cell. In certain embodiments, the immunity modulator protein is trans to the bacteriocin mononucleotide. If the immunity modulator activity of the microbial cells is not desired (e.g., if a virus is lost or if an environmental condition causes a decrease in immunity modulator activity), then it can be lost. The bacteriocin activity can be induced by both the microbial cells and other strain cells.

“In some embodiments, it is possible to control a ratio between two or more microbial strains or species. FIG. 3 illustrates an example control of ratios. FIG. 3 (see cells 300, 310). In some embodiments, a first microbial strain or species loses an immunity modulator activity via any of the mechanisms discussed herein when it is less desired than a bacteriocin-producing second strain or species, increasing the ratio of second strain or species to the first strain or species. A bacteriostatic or bacteriocin is used to control the ratio between a first and second strain/species. This allows for the control of growth to be easily reversed and/or minimizes the chance of either strain/species being eliminated. A first microbial species or strain can produce a first type of bacteriocin when it is stimulated by a promoter. This could be an intermediate or an industrially useful product. The levels of the bacteriocin will increase with increasing levels of the compound of concern. In some cases, the second microbial species or strain that produces the compound of interest (or catalyzes its production) may not be immune modulator active for the bacteriocin. The bacteriocin levels increase with increasing levels of the compound/substance of interest. This neutralizes the second strain, which lacks the appropriate immunity modulator or has insufficient immunity modulator activity to protect against the effects of the bacteriocin. The relative levels of the first and second strains increase. A first microbial strain may produce a first product with first bacteriocin activities, while a second strain will produce a second product with second bacteriocin activities. In some embodiments the first product and second product can be intermediates in the biosynthetic pathway. The first microbial strain may provide both a first and a second immunity modulator activitiy. In this case, the second immunity modulator activity could protect against the second Bacteriocin. However, the accumulation of the first product can negatively regulate the second immunity modulator activity (e.g. The presence of the first product can suppress expression of the second immune modulator, while the first immunity modulator activity may protect against the first. A second microbial strain may also be capable of providing a first and second immune modulator activity. However, the accumulation of the second product can negatively regulate the first immunity modulator activity (e.g. The presence of the second product can repress the expression of the first immune modulator. When a high level of the first product is present, the second immunity modulator of the first microbial species is activated and the microbial cells from the first strain are neutralized with the second bacteriocin. This increases the ratio of second strain to first strain and increases the relative amount second product to first. The second immunity modulator in a second microbial strain can be inactivated if a large amount of second product is accumulated. In this case, the microbial cells in the second strain will be neutralized by the first bacteriacin. This increases the ratio of first and second strains and decreases the amount of second product. Depending on the product level, the ratio between the first and second stain can be adjusted. In some embodiments, the ratios of first and second strains can be maintained in an equilibrium. In some embodiments, the equilibrium of ratios between the first and second products is maintained. In some embodiments, the second immunity modulator of the first microbial species responds to a first environment condition or compound. The ratio between the second and first microbial strains is controlled in the same way as above. In some embodiments the second microbial species’s first immunity modulator responds in a second environment condition or compound. The ratio between the first microbial strain and the second is controlled as above.

“In some embodiments it is desirable that a microbial cells be contained within a specific environment. For example, so that the first microbial cells can survive only in a certain culture medium like industrial feedstock. A microbial cell may contain a bacteriocin and an immunity modulator polynucleotide. In some cases, the immunity modulator corresponds with the bacteriocin. Some embodiments show that when the microbial cells are in a desired environment, they produce an active bacteriocin, and the corresponding immunity modulator. However, when the microbial cells escape the desired environment the active bacteriocin is produced but not the active immunity modulator. The microbial cells can be grown in any environment they choose, but are neutralized by their own bacteriocin if it escapes. In some embodiments, for example, the bacteriocin encoded in the bacteriocin mononucleotide can be expressed constitutively, while the immunity modulator can only be expressed when the microbial cells are in a desired environment. In some embodiments, for example, the bacteriocin encoded in the bacteriocin mononucleotide can be constitutively expressed while the immunity modulator can only be expressed when the microbial cells are in a desired environment. In some embodiments, the immune modulator’s transcriptional activator can only be present in the desired environment. In some embodiments, for example, the bacteriocin encoded in the bacteriocin mononucleotide as well as the immunity modulator are constitutively expressed. However, if the microbial cells escape, the immunity modulator can be deleted using the FLP-FRT method. It is possible that a genetic system to neutralize an escaped microbial cells is not used in the culture. This can lead to mutations that reduce or eliminate the functionality of the genetic system. If the microbial cell escapes, it is possible that the genetic system may cease to function. It is, however, appreciated that a bacteriocin/immunity modator system can be used both inside and outside of a culture to control growth of genetically engineered cells and/or neutralize invading microorganisms. Genetic drift can be minimized by using selective pressure according to some embodiments. This selective pressure can be used in accordance to some embodiments to help ensure that, if a microbial cell escapes from the desired environment, the bacteriocin/immunity module system will function to neutralize it. In some embodiments, a single genetically engineered circuit (e.g. a bacteriocin/immunity module system) can be used to both neutralize other microbial cultures in a desired environment and to further neutralize a microbial cell or its clones after they escape from that environment. According to some embodiments, it is possible to adjust the configurations of bacteriocins described herein so that the escaping microbial organism can be neutralized by its own bacteriacins and/or the bacteriocins from its direct or indirectly progeny and/or the bacteriocins in the cell of another escaped cell or its direct or indirect parent.

“Some embodiments allow a microbial cells to control growth in more than one way. A microbial cell may perform more than one function in some embodiments. In some cases, the microbial cells use the same bacteriocin/immunity moduleator pair for multiple functions. Some embodiments use a bacteriocin/immunity moduleator pair to perform a first function and another bacteriocin/immunity modator pair to perform a second function. In some embodiments, a microorganism can express a bacteriocin that limits the growth of non-expressed? bacteria. Clones that have lost immunity modulator activity can be used to provide containment in the desired environment. However, if the microbial cells are not in the desired environment, they may still express bacteriocin. FIG. illustrates a schematic illustration of these two forms of growth regulation. 2. In some embodiments, the first microbial cells can express a bacteriocin that inhibits the growth of the second microbial cells and can neutralize invading cells. FIG. 2 illustrates a schematic illustration of these two forms of growth regulation. 3. Some embodiments provide growth control using two or more bacteriocin immunity modulator pairs. In some cases, each type of growth control can be provided with a different pair of bacteriocin immunity modator pairs. A plasmid can have a first immune locus that encodes a desired product. A first bacteriocin will neutralize a clone that has lost the plasmid. The second immunity modulator polynucleotide, which corresponds to a second immune modulator, can be integrated into a microbial cell’s genome and can be silenced if the microbial cells escape from their desired environment. For example, the second immunity modator polypeptide can be found in an FLP-FRT cassette. This cassette is then excised after escape. The second bacteriocin can neutralize the microbial cells upon escape.

“It should be noted that some embodiments herein are compatible avec poison-antidote system. In some embodiments, a microbial cells, along with a bacteriocin or immunity modulator, also includes a poison-antidote device that kills or arrests the cell when it’s not in a desired environment.

“It is possible to control the growth and development of multiple types of microbial cell. An environment could contain, or potentially include, multiple types of undesirable microbial organisms. Different microbial organisms may be more susceptible to bacteriocins than others (for example, because they have different immunity modulators), so a mixture of two or more of bacteriocins is possible (e.g. A?cocktail? A?cocktail? (a mixture of bacteriocins and other microbial organisms) is useful in controlling their growth. One microbial cell may produce two or more bacteriocins. In some cases, this includes at least 2, 3, 5, 6, 7, 8, 9, 13, 14, 15, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or 50 different types of bacteriocins. In some embodiments, a mixture of two or more different bacteriocin-producing microbial cells are provided, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 different bacteriocin-producing microbial cells, including ranges between any two of the listed values. Optionally, one or more of the bacteriocin-producing microbial cells can produce two or more different bacteriocins.”

Summary for “Controlled Growth of Microorganisms”

Since the dawn of time, humans have used microbial organisms for product generation, such as in the production of cheese, beer and wine. Over the centuries, microbial organisms-mediated processes were studied and scaled up, often by controlling fermentation conditions, or identifying phenotypic traits of microbial bacterias.

Many products are currently made using microbial organisms. Microbial organisms can be grown in controlled, sterile environments in laboratories and pharmaceutical manufacturing processes. However, feedstocks that are used in various industrial processes that involve microorganisms may not be sterile and could contain many strains or species. Genetically engineered microorganisms are not suitable for industrial processes such as those that involve feedstocks, or are exposed to microorganisms in their environment, which could potentially contaminate the culture, and may also include changing environmental conditions. These microorganisms have been designed to manage their own growth, that of other microorganisms, and/or respond to environmental changes. These microorganisms can be grown in non-sterile and less rigidly controlled feedstocks. These microorganisms are useful in the production of consistent, robust products across a variety of environments and feedstocks.

“One embodiment of this invention includes a first cell that contains a nucleic acids encoding a secreted Bacteriocin. This nucleic acids controls the growth of another microbial cells. A second cell is also provided with a nucleic Acid which confers resistance. The first cell has been genetically engineered so that the activity or expression of the nucleic a which confers resistance can be controlled. This embodiment states that the activity or expression of the nucleic acids which confer resistance to the bacteriocin can be reduced to a point where the first microbial cells are neutralized by the bacteriocin. Some aspects of this embodiment state that the first microbial cells have been genetically engineered in order to produce a desired product. Some aspects of this embodiment state that the secreted Bacteriocin has been chosen to maintain at least one environment in a culture where the desired product is being produced by the first microbial cells. Some aspects of this embodiment state that the culture contains at least one invading microorganism. Some aspects of this embodiment state that at least one condition of the cultivation is controlling the growth of the second microorganism. The second microbial organism may be a common contaminate in the culture. Some aspects of this embodiment state that the second microbial cells are different species, genuses, or strains from the first. Some aspects of this embodiment state that the microbial cells also contain a nucleic acids encoding a second secreted Bacteriocin. This nucleic is responsible for controlling the growth of a third cell. Also, it confers resistance against the second bacteriocin. The first microbial cells have been genetically engineered to regulate the activity or expression of the nucleic. The bacteriocin kills a second microbial cells according to certain aspects of this embodiment. Some aspects of this embodiment state that the bacteriocin decreases the growth rate for the second microbial cells. Some aspects of this embodiment state that the bacteriocin stops the growth of the second microorganism. A regulatable promoter controls transcription of the nucleic acids conferring resistance to the Bacteriocin, according to certain aspects of this embodiment. Some aspects of this embodiment state that the activity of the polypeptide encoded in the nucleic acids conferring resistance to the Bacteriocin can be controlled. Some aspects of this embodiment state that the nucleic acids encoding the Bacteriocin are on the chromosomes of the microbial cells. Some aspects of this embodiment state that the nucleic acids conferring resistance to the Bacteriocin are on a plasmid. Some aspects of this embodiment state that the nucleic acids encoding the Bacteriocin are on the chromosomes of microbial cells, while the nucleic acids conferring resistance to the Bacteriocin are on plasmids. Some aspects of this embodiment state that the nucleic acids encoding the Bacteriocin and those conferring resistance to the Bacteriocin are located on one or more different plasmids. Some aspects of this embodiment state that the first microbial cells are selected from among bacteria, yeast, or algae.

“Another embodiment described herein comprises a method for controlling the growth a second microbe in a culture medium. This method involves the cultivation of a first microbial cells in conditions where the first microbial cells produce bacteriocin sufficient to control growth of the second microbe. Some aspects of this embodiment require that the culture be maintained continuously for at least 30 consecutive days. This could include at least 30, 35-40, 45, 55, 60 and 65, 70. 75, 85, 95, 90. 95, 100. 110, 130, 140. 150, 160. 170. 180. 190. 200. 250. 350. 400. 450. The method also includes the detection of at least one change within the culture medium. This change may include the presence or an increase in activity of a third cell. Reengineering the first cell to produce a second form of bacteriocin in response to this change is necessary to control growth of the third cell.

“Another embodiment is disclosed that allows for the detection of a presence, absence or amount of a chemical in a culture. This can be done by culturing a first genetically-engineered microbial cells that contain a bacteriocin and a genetically regulateable promoter. The regulatable promor controls transcription so that (a) transcription is driven by the regulatable promotor in the presence of a molecule but not in its absence; (b) transcription is driven in the absence but not in its presence. This can include determining the amount of genomic nucleic acids in the first microbial cells from the culture. This can include determining the presence, absence, and quantity of a particular nucleic acids sequence characteristic of the first microbe. The method can also be used to compare the amount of the nucleic acids sequences characteristic of the first microbial cells to the quantity of a reference sequence.

“Another embodiment described herein comprises a genetically engineered virus that contains a nucleic acids conferring resistance against bacteriocin. In this case, the activity or expression of the nucleic acids is modified to respond to the presence, concentration, or absence of a component in a feedstock. The vector may also include a nucleic acids that encode the bacteriocin, according to certain aspects. The vector may also contain a nucleic acids that encode a product, according to certain aspects of this embodiment.

“Another embodiment described herein is a kit that can include a plurality strains of genetically engineered microorganisms. Each strain has been genetically modified to allow expression or activity of a nucleic acids which confer resistance to a different type of bacteriocin to being regulated.

“Another embodiment described herein involves a method for identifying at minimum one bacteriocin that modulates the growth in at least 1 microbial cells in an industrial culture media. This includes contacting the medium with a composition or medium containing the at most one bacteriocin and determining if the at-least one bacteriocin has any desired effect on the growth in the at-least one microbial cells. The method, according to certain aspects of this embodiment, involves contacting the industrial medium with at least one bacteria produced by a first microbial cells as described herein. Some aspects of this embodiment state that the at least one bacteriocin created by the first microorganism is contained in the supernatant from the culture containing the first microbial cells. The method also includes the creation of a genetically engineered microorganism to produce at most one bacteriocin that has been shown to have a desired effect upon the growth of at least one of the microbial cells. Some aspects of this embodiment state that the least one microbial cells is an organism that is a common invader in the industrial culture medium. Some aspects of this embodiment state that the least one microbial cells is an organism that has invaded an industrial culture.

“Another embodiment described herein involves a system for neutralizing unwanted microbial organisms within a culture medium. The system may include a first environment that includes a culture medium and a second environment that contains a second microorganism that secretes two or three different bacteriocins. In this second environment, immunity modulators are provided for each of these bacteriocins. The second environment is physically isolated from the first environmental so that the second organism cannot move between the environments. The system can also include a first microorganism in the culture medium. This means that the first microbial species does not secrete any of the two or three different bacteriocins and the first microbial entity is not neutralized by any one of the two or three different bacteriocins. This embodiment states that the first microbial organism is not genetically modified. Some aspects of this embodiment state that the first microbial organism ferments an ingredient of the culture medium. Some aspects of this embodiment state that the culture medium is decontaminated by the first microbial organism. Some aspects of this embodiment state that the first microbial organism performs photosynthesis. The substrate used in the photosynthesis is also included. Some aspects of this embodiment state that the second environment is separated by the first environment using at least one of a membrane or mesh or filter. However, it is not permeable for the second microbial species. Some aspects of this embodiment state that the second microbial organism secretes at most three bacteriocins. These include at least 3, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, and 20 bacteriocins. Some aspects of this embodiment state that the second environment contains at least one third microbial organism. This organism also secretes some bacteriocins. Some aspects of this embodiment state that the third microbial organism secretes at most 2 bacteriocins. For example, at least 2, 3, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 19 or 20 bacteriocins. A method for storing a feedstock is another embodiment described herein. This could include providing a feedstock and providing a first microorganism that secretes two or three different bacteriocins. Then, the method involves contacting the feedstock with these bacteriocins and then storing it for the desired time. According to certain aspects of this embodiment contact the feedstock using the bacteriocins means that the feedstock is contacted with the microbial organism. Some aspects of this embodiment state that contacting the feedstock using the bacteriocins involves putting the microbial species in fluid communication with it, while keeping the feedstock and microbial organisms physically separated. This ensures that the bacteriocins can contact the feedstock but not the microbial. Some aspects of this embodiment state that the separation is maintained by at minimum one or more membranes, meshes, filters, or valves that are permeable to two or more different types of bacteriocins but not the first microbial species. The method may also include fermenting the feedstock using a second microbial organism before or concurrently with contact with the bacteriocins. Some aspects of this embodiment state that the fermentation can be used to produce a desired component or remove an undesirable component from the feedstock. Some aspects of this embodiment state that the desired time period is at least one month. This could be one, two or three, four, five to six months, seven to eight weeks, nine to nine, ten, eleven, twelve, or twelve. Some aspects of this embodiment state that the desired time period is at least six months. This could be six, seven or eight, nine, ten, eleven, twelve, or twelve. Some aspects of this embodiment state that the first microbial organism must secrete at least three bacteriocins. These could include at least 3, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

“Genetically engineered microbial organisms can be found in some of the examples. Some embodiments of the microbial organisms can be engineered to limit the growth of the microbial populations in environments such as those that use a feedstock. As used herein, ?neutralizing? Activity (and variations on the same root word), can refer to either arresting microbial reproduction or cytotoxicity. It is possible to engineer microbial organisms to produce bacteriocins. These secreted polypeptides can neutralize microorganisms. Certain bacteriocins can be repelled by microbial organisms that have bacteriocin immunity modators. In some embodiments, the first microbial organism is designed to secrete bacteriocins. Some embodiments select the bacteriocins based on the type and location of the microbial cells, the composition of the medium or the geographic location. This is to target specific contaminating organisms that are associated with the medium or geographical location. Other microbial species that exhibit desired characteristics can produce bacteriocin immune modulators and thus survive in the presence bacteriocins. Unwanted microbial species (for example, contaminants, organisms that have lost a desired characteristic, or organisms involved in an industrial process, but whose growth or production is not desirable under the prevailing conditions), fail to produce bacteriocin immuno modulators and are therefore neutralized by the Bacteriocins.

“Microbial Organisms”

“Genetically engineered microorganisms can be provided according to certain aspects. Genetically engineered microbial organism is used herein. ?microorganism,? These root terms can be used in various variations (such as pluralizations) and include genetic modification of any naturally occurring species, fully synthetic prokaryotic and eukaryotic unicellular species as well as Archae species. This expression can be used to refer to cells of bacteria, fungal, and algae.

Examples of microorganisms that could be used according to the embodiments are, but not limited to, yeast, bacteria, and algae. Full synthetic microorganism genomes are also possible to be synthesized and transferred into single microbial cell, to create synthetic microorganisms that can self-replicate continuously (see Gibson et. al. (2010), “Creation of Bacterial Cells Controlled by Chemically Synthesized Genes”, Science 329: 52?56. This article is hereby included by reference in its entirety. In some cases, the microorganism can be fully synthesized. The desired combination of genetic elements can be assembled onto a chassis to create a fully or partially synthetic microorganism. Wright et al. also provide a description of genetically engineered microorganisms for industrial purposes. (2013). Building-in biosafety to synthetic biology. Microbiology 159: 1221-1235.”

“A variety of bacterial strains and species can be used in accordance to embodiments herein. Genetically modified variants or synthetic bacteria based upon a?chassis?” An example of a species is provided. Exemplary bacteria that can be used to make industrially useful characteristics according to the embodiments are, among others, Bacillus species (for instance Bacillus subtilis and Bacillus), Streptomyces species and Streptomyces species.

“A variety can be used to make yeast strains and species, including genetically modified varieties and synthetic yeast that is based on a?chassis. An example of a species is provided. Exemplary yeast can be provided with industrially-applicable characteristics.

“A variety can be used to create synthetic algae using genetically modified strains or species based on a ‘chassis. It is possible to create a new species. Some embodiments of the algae include photosynthetic microalgae. Examples of algae species that could be useful in biofuels and can be used according to embodiments herein include Botryococcus braunii and Chlorella species. Many algae species can also be used for fertilizer products, food production, waste neutralization, environmental remediation, carbohydrate manufacturing (for instance, biofuels).

“Bacteriocins”

“As used herein, ?bacteriocin,? This root term and variants of it, refers to a protein that is secreted in host cells and can neutralize at most one other cell than the host cell in which the polypeptide was made. As used herein, ?bacteriocin? Also includes a cell-free version or chemically synthesized form of this polypeptide. A cell that expresses one particular immune modulator. A cell that expresses a specific?immunity modulator? is immune to the neutralizing effect of a particular bacteriocin (or group of bacteriocins). The bacteriocins are able to neutralize the effects of bacteriocins on a cell that produces it and/or other microbial organisms, provided they don’t produce an immunity modulator. A host cell can secrete bacteriocins to inhibit growth or cytotoxicity in order to affect a variety of microbial organisms. The translational machinery (e.g. A ribosome, etc. A microbial cell. A bacteriocin can be chemically synthesized in some embodiments. A polypeptide precursor can be used to make some bacteriocins. To obtain the bacteriocin polypeptide, the precursor polypeptide can be cleaved (for example by a protease). In some embodiments, the precursor polypeptide is used to make a bacteriocin. A bacteriocin may be a polypeptide which has been subject to post-translational modifications such as cleavage or addition of one or more functional group.

“?Antibiotic,? “?Antibiotic” and other variations of this root term refer to a metabolite or intermediate in a metabolic pathway that can kill or arrest growth of at least one microbe. Microbial cells can produce some antibiotics, such as bacteria. Some antibiotics can also be made chemically. It is clear that antibiotics and bacteriocins can be synthesized chemically.

“Neutralizing activity can be described as the arrest of microbial reproduction or cytotoxicity. Some bacteriocins can be cytotoxic (e.g. ?bacteriocide? ?bacteriocide? Some bacteriocins may inhibit the reproduction of microbial species (e.g. ?bacteriostatic? ?bacteriostatic?

“It should be noted that there have been non-bacteriocin strategies to target different microbial organisms. KAMORAN is one example. A chemical has been suggested to target Lactic Acid Bacteria family bacteria (LAB) (see Union Nationale des Groupements de Distillateurs D’Alcool (2005)?Kamoran). It should be noted that phage was also proposed to target LAB-family bacteria (see U.S. Pub. No. 2010/0330041). It should be noted that pesticides are being considered to target a variety of contaminating microorganisms (see McBride and al.,?Contamination management in low cost open algae ponds for biofuels production?). Industrial Biotechnology 10: 221-227 (2014). However, bacteriocins have many advantages over pesticides and phages. Bacteriocins are able to prevent potentially toxic runoff from a feedstock. Bacteriocins may have a higher effectiveness against certain undesired microorganisms than chemicals, pesticides or phages. Logarithmic growth can produce bacteriocins, which can easily be scaled up or down. This is in contrast to phages and chemical/pesticide system, which can be less scalable. Bacteriocins, for example, can be used to control which organisms are neutralized. This is useful for avoiding the neutralization of industrially valuable microbial organisms in culture media. Phage production at industrial scale can be challenging. They can also be very difficult to control. However, bacteriocins according to some embodiments can be part of an industrial process. They can contain gene and/or control a fermenting process via bacteriostatic activities. Immunity control can also be used to adjust the susceptibility of microorganisms involved in an industrial process. Bacteriocins are typically low in toxicity for industrial uses such as food for animals or humans. It is possible that bacteriocins according to some embodiments herein could be used as a food additive.

“In some embodiments, a specific neutralizing activity (e.g. “Some embodiments have a particular neutralizing activity (e.g., arrest of microbial replication) that is determined by the type of microbial regulation desired. In some cases, microbial cells can be engineered to express a particular combination or bacteriocins. In some embodiments, for example, microbial cells can be engineered to express specific bacteriocins depending on how they are regulated. For example, if the contaminating cells must be killed, at least one cytotoxic-bacteriocin may be provided in some embodiments. One or more bacteriocins or combinations thereof that are effective against contaminants found in particular cultures, geographic locations, or particular types of culture grown in particular geographical areas may be used in some embodiments. A bacteriocin which inhibits microbial reproduction may be used in some embodiments. Many bacteriocins have the ability to neutralize microbial organisms, regardless of their ecological niche. In some cases, it is desirable to have a specific spectrum of bacteriocin activities. To this end, a bacteriocin may be selected from a host that is similar or identical to the microbial organism/s being targeted by the bacteriocin.

“In some embodiments, one, or more, bacteriocin activities may be selected before culture growth and one or two microbial organisms are engineered for the desired culture environment. Some embodiments allow for bacteriocins to be chosen based on their ability neutralize invading organisms that are likely to try to grow in the culture. Another embodiment is where strain A produces intermediate A and strain B converts it into intermediate B. In this case, the equilibrium can shift to favor strain B through the generation of a profile of bacteriocin activities that favors strain A. A lack of intermediateA will cause the equilibrium profile to favor strain A. This profile will inhibit or prevent growth of strain B. One or more bacteriocin activities can be selected depending on the conditions of an existing culture. If certain invaders are found in a culture environment, the?neutralizer’ is selected. It is possible to engineer microorganisms to produce bacteriocins in order to neutralize identified invaders. Some embodiments add genetically engineered cells capable of producing bacteriocins to an existing culture to re-equilibrate it, such as when a certain microbial type grows in the microbial culture. Some embodiments add genetically engineered cells that make bacteriocins to an existing culture to neutralize all, or substantially all, of the microbial cells within the culture. This is done to, for example, to remove an industrial culture from the culture environment to allow for the introduction of a new industrial culture.

“For example, some embodiments may have anti-fungal activity (such anti-yeast activity). There are many bacteriocins that have anti-fungal activity. For example, bacteriocins from Bacillus have been shown to have neutralizing activity against yeast strains (see Adetunji and Olaoye (2013) Malaysian Journal of Microbiology 9: 130-13, hereby incorporated by reference in its entirety), an Enterococcus faecalis peptide (WLPPAGLLGRCGRWFRPWLLWLQ SGAQY KWLGNLFGLGPK, SEQ ID NO: 1) has been shown to have neutralizing activity against Candida species (see Shekh and Roy (2012) BMC Microbiology 12: 132, hereby incorporated by reference in its entirety), and bacteriocins from Pseudomonas have been shown to have neutralizing activity against fungi such as Curvularia lunata, Fusarium species, Helminthosporium species, and Biopolaris species (Shalani and Srivastava (2008) The Internet Journal of Microbiology. Volume 5, Number 2. DOI: 10.5580/27dd?accessible on the worldwide web at archive.ispub.com/journal/the-internet-journal-of-microbiology/volume-5-number-2/screening-for-antifungal-activity-of-pseudomonas-fluorescens-against-phytopathogenic-fungi.html#sthash.d0Ys03UO.1DKuT1US.dpuf, hereby incorporated by reference in its entirety). Botrycidin AJ1316 is an example (see Zuber, P. et al. (1993) Peptide Antibiotics. In Bacillus subtilis and other Gram-Positive Bacteria : Biochemistry, Physiology and Molecular Genetics ed Sonenshein et., pp. 897-916, American Society for Microbiology. hereby incorporated in its entirety). and Alirin B1 (see Shenin, et al. (1995) Antibiot Khimioter 51: 3-7. Antifungal activities have been demonstrated in B. subtilis. In some cases, such as those that require neutralization of a fungal organism, the bacteriocin may contain at least one botrycidin A1316 or alirin C1.

“For example, some embodiments make it desirable to have bacteriocin activity within cyanobacteria cultures. Some embodiments provide bacteriocins to neutralize cyanobacteria. Some embodiments provide bacteriocins to kill invading microorganisms that are commonly found in cyanobacteria cultures. A wide range of cyanobacteria species have been found to contain clusters of conserved polypeptides of bacteriocin. Wang et. al. reported that at least 145 clusters of putative bacteriocin genes have been found in at least 43 species of cyanobacteria. Genome Mining demonstrates the widespread occurrence of gene clusters coding bacteriocins within Cyanobacteria. PLoS ONE 6(7) : e22384, hereby incorporated in its entirety by reference. Table 1.2 shows exemplary cyanobacteria Bacteriocins. These are the SEQ ID NO’s 422, 422, 426, 428 and 30.

“In some embodiments, the host cells themselves are a microbial cell. Bacteriocins can neutralize cells from a different strain or species than the host cell in some embodiments. If the cells are not equipped with an immune modulator, some embodiments allow bacteriocins to neutralize cells of the same strain or species as the host cells. The skilled artisan will be able to tell that bacteriocins are capable of neutralizing both host and non-host microorganisms. Bacteriocins are capable of neutralizing cells other than those in which they were produced. This means that poison molecules can only be killed the specific cell in which they were produced.

“A variety of bacteriocins were identified and characterized. Exemplary bacteriocins may be classified as “class I” without being restricted by any particular theory. bacteriocins that are subject to post-translational modifications. bacteriocins which are usually unmodified. Furthermore, the exemplary bacteriocins from each class can be classified into different subgroups. Table 1.1 is adapted by Cotter, P. D., and others. Are bacteriocins a viable alternative to antibiotics. Nature Reviews Microbiology 11 (95-105) is hereby included by reference in its entirety.

“Bacteriocins are not limited by any one theory. They can neutralize a target microbial cells in many ways.” A bacteriocin, for example, can penetrate a cell wall to depolarize it and interfere with respiration.

“TABLE 1.1\nClassification of Exemplary Bacteriocins\nGroup Distinctive feature Examples\nClass I (typically modified)\nMccC7- Is covalently attached MccC7-C51\nC51-type to a carboxy-\nbacteriocins terminal aspartic acid\nLasso peptides Have a lasso structure MccJ25\nLinear azole- or Possess heterocycles MccB17\nazoline-containing but not other\npeptides modifications\nLantibiotics Possess lanthionine Nisin,\nbridges planosporicin,\nmersacidin,\nactagardine,\nmutacin 1140\nLinaridins Have a linear Cypemycin\nstructure and contain\ndehydrated amino acids\nProteusins Contain multiple Polytheonamide\nhydroxylations, A\nepimerizations and\nmethylations\nSactibiotics Contain sulphur-?- Subtilosin A,\ncarbon linkages thuricin CD\nPatellamide-like Possess heterocycles Patellamide A\ncyanobactins and undergo\nmacrocyclization\nAnacyclamide- Cyclic peptides consisting Anacyclamide\nlike of proteinogenic amino A10\ncyanobactins acids with prenyl\nattachments\nThiopeptides Contain a central pyridine, Thiostrepton,\ndihydropyridine or nocathiacin\npiperidine ring as I, GE2270 A,\nwell as heterocycles philipimycin\nBottromycins Contain macrocyclic a Bottromycin A2\nmidine, a decarboxylated\ncarboxy-terminal\nthiazole and carbon-\nmethylated amino\nacids\nGlycocins Contain S-linked Sublancin 168\nglycopeptides\nClass II (typically unmodified or cyclic)\nIIa peptides Possess a conserved Pediocin PA-1,\n(pediocin YGNGV motif enterocin\nPA-1-like (in which N represents CRL35,\nbacteriocins) any amino acid) carnobacteriocin\nBM1\nIIb peptides Two unmodified ABP118,\npeptides are required lactacin F\nfor activity\nIIc peptides Cyclic peptides Enterocin AS-48\nIId peptides Unmodified, linear, MccV, MccS,\nnon-pediocin-like, epidermicin NI01,\nsingle-peptide bacteriocins lactococcin A\nIIe peptides Contain a serine-rich MccE492, MccM\ncarboxy-terminal\nregion with a non-ribosomal\nsiderophore-type modification”

“Numerous bacteriocins may be used according to the embodiments. Table 1.2 shows examples of bacteriocins. In certain embodiments, at most one bacteriocin is comprised of a sequence of polypeptides from Table 1.2. Some bacteriocins are able to function as pairs of molecules, as shown in Table 12.2. It will be clear that, unless stated otherwise, functional bacteriocins are essentially a pair of molecules. If a functional bacteriocin is used, or if it provides a bacteriocin? The like is discussed herein. Functional bacteriocin pair are included alongside bacteriocins which function separately. Refer to Table 1.2 for information on?organisms that are of origin? The alternative names and/or strain information of organisms that produce the indicated bacteriocin are listed in parentheses.

“Embodiments” herein include peptides as well as proteins that are identical to the bacteriocins listed in Table 1.2. “Identity” is a broad term that can be used to describe any combination of nucleic acid or protein sequence homology, or three-dimensional homology. The term “identity” can refer to nucleic acids, protein sequence homology, or three-dimensional homology. There are many methods that can be used to determine the nucleic acid sequence homology or three-dimensional homology of polypeptides. These techniques are used to determine the degree of identity between a sequence, domain or model and a target sequence or domain. There are many functional bacteriocins that can include features of the bacteriocins described herein. This allows for a wide range of identity to the bacteriocins listed in Table 1.2. A bacteriocin may have at least 50% identity in some embodiments. For example, 51%/52%/53%/54%/56%/57%/58%/69%/70%/74%/76%/77%/78%/78%/79%/78%/79%/78%/79%/79%/80%, 80%, 85%, 85%, 86%/87%, 89%, 89%, 99%, 91% 92% 93% 94% 95% 96% 97% 98%, 98%, 99.9% identity to any of Table 1.2. BLAST software (Altschul S. F. et al.) can be used to determine percent identity. (1990)?Basic Local Alignment Search Tool. J. Mol. Biol. 215:403-410, accessible on the world wide web at blast.ncbi.nlm.nih.gov) with the default parameters.”

“In some embodiments, the polynucleotide that encodes a bacteriocin according to this invention is provided. The polynucleotide may be contained within an expression vector in some embodiments. In some embodiments, the expression vector or polynucleotide is found in a microbial cells. Table 1.2 shows examples of polynucleotide sequences that encode the polypeptides. Table 1.2 shows examples of polynucleotides that are based on reverse translation. SEQ ID NOS: 341 to 419 (odd numbers SEQ ID) are examples. A skilled artisan will quickly understand that a polypeptide can be encoded by more than one polynucleotide. The genetic code can be degenerate and codon usage may vary depending on the organism where the gene product is expressed. A polynucleotide that encodes a bacteriocin may be selected according to the codon usage of the organism that possesses the gene product. A polynucleotide that encodes bacteriocin may be codon optimized according to the specific organism that carries it.

“While Table 1.2 contains bacteriocins that are naturally occurring, the skilled artisan will recognize that there are variants of these bacteriocins, naturally-occurring Bacteriocins, and variants thereof. Synthetic bacteriocins may also be used in some of the embodiments. These variants may have a higher or lower level of cytotoxic activity or growth inhibition activity than the wild type protein in some embodiments. Many motifs have been identified as characteristics of bacteriocins. The N-terminal consensus sequence of class IIa Bacteriocins is represented by the motif YGXGV (SEQID NO: 2), in which X can be any amino acid residue. In some embodiments, the N-terminal sequence of a synthetic bacteriocin is at least 50% identical to SEQID NO: 2. A synthetic bacteriocin may include a N-terminal sequence that includes SEQ ID NO. 2. Some class IIb bacteriocins also contain a GxxxG motif. Without being limited by any particular theory, it is believed that the GxxxG motif can mediate association between helical proteins in the cell membrane, for example to facilitate bacterioncin-mediated neutralization through cell membrane interactions. In some embodiments, the motif in bacteriocin facilitates interaction with the cell membrane. The bacteriocin may contain a GxxxG motif in some embodiments. A helical structure can be added to a bacteriocin containing a GxxxG-motive. This structure is not the only one that is described in this article. Structures that have substantially the exact same effect on microorganisms as any of the bacteriocins provided herein are also included in the definition of?bacteriocin.

“As used herein ?bacteriocin polynucleotide? Refers to a polynucleotide that encodes a bacteriocin. In some embodiments, at least one bacteriocin is present in the host cell.

“Bacteriocin immunity modulators”

“Table 2 shows examples of bacteriocin-based immunity modulators. The immunity modulators shown in Table 2 are natural-occurring. However, a skilled artisan will recognize that there are other naturally-occurring immunity modators than those listed in Table 2. Synthetic immunity modulators or variants can also be used.

“In certain embodiments, an immunity modulator, or combination thereof, confers immunity against a specific bacteriocin or class or category of Bacteriocins or a particular combination of Bacteriocins. Table 2 lists examples of bacteriocins for which immunity modulators may confer immunity. Table 2 lists the ‘organism of origin? These immunity modulators are exemplary examples. However, they can be easily expressed in other naturally occurring, genetically modified or synthetic microorganisms to produce the desired bacteriocin activity, in accordance with certain embodiments. As such, the term “immunity modulator” is used herein. This term refers to not only structures described herein but also to structures that have substantially similar effects to the?immunity modator? Structures described herein include fully synthetic immunity modulators and immunity modulators which provide immunity to bacteriocins functionally equivalent to those disclosed herein.

Table 2 shows examples of polynucleotide sequences that encode the polypeptides. Table 2 lists the examples of polynucleotide sequences that encode the polypeptides. A skilled artisan will quickly understand that the genetic code can be degenerate. A polynucleotide that encodes a bacteriocin immune modulator may be selected according to the codon usage of an organism expressing this gene. A polynucleotide that encodes a bacteriocin immune modulator may be codon optimized according to the specific organism that possesses the modulator. There are many functional immunity modulators that can include features from the immunity modulators described herein. This allows for a wide range of combinations, which provides a high degree of identity to the immunity modators listed in Table 2. An immunity modulator may have at least 50% identity in some embodiments. For example, 51%-52%, 53%-54%, 56%-57%, 58%-59%, 69%-69%, 70%, 71%-72%, 74%-73%, 74%-76%, 77% and 78%, 77%-77%, 78%-79%, 78%-79%, 78%-78%, 79%-79%, 80% or 90% identity to any of the polypeptides listed in Table 2.

“TABLE 2\nExemplary bacteriocin immunity modulators\nPoly- Poly-\npeptide nucleotide\nSEQ Polypeptide Organism SEQ\nID NO: Name Sequence of origin ID NO: Polynucleotide Sequence\n452 Microcin MSYKKLY Escherichia 453 ATGAGTTATAAAAAAC\nH47 QLTAIFSLP coli TGTACCAATTGACGGCT\nimmunity LTILLVSLS ATATTTAGTTTACCTCT\nmodulator SLRIVGEG TACTATCTTATTGGTTT\nMchI NSYVDVFL CACTTTCATCCCTTCGG\nSFIIFLGFIE ATTGTTGGCGAAGGGA\nLIHGIRKIL ATTCTTATGTTGACGTT\nVWSGWKN TTTCTAAGCTTTATAAT\nGS ATTTCTTGGTTTTATTG\nAGCTGATTCATGGGATT\nCGAAAGATTTTGGTCTG\nGTCAGGCTGGAAAAAC\nGGAAGTTAA\n454 Colicin-E3 MGLKLDLT Escherichia 455 ATGGGACTTAAATTGG\nimmunity WFDKSTED coli ATTTAACTTGGTTTGAT\nmodulator FKGEEYSK AAAAGTACAGAAGATT\n(Colicin-E3 DFGDDGSV TTAAGGGTGAGGAGTA\nchain B) MESLGVPF TTCAAAAGATTTTGGAG\n(ImmE3) KDNVNNG ATGACGGTTCAGTTATG\n(Microcin- CFDVIAEW GAAAGTCTAGGTGTGC\nE3 VPLLQPYF CTTTTAAGGATAATGTT\nimmunity NHQIDISD AATAACGGTTGCTTTGA\nmodulator) NEYFVSFD TGTTATAGCTGAATGG\nYRDGDW GTACCTTTGCTACAACC\nATACTTTAATCATCAAA\nTTGATATTTCCGATAAT\nGAGTATTTTGTTTCGTT\nTGATTATCGTGATGGTG\nATTGGTGA\n456 Colicin-E1 MSLRYYIK Escherichia 457 ATGAGCTTAAGATACTA\nimmunity NILFGLYC coli CATAAAAAATATTTTAT\nmodulator TLIYIYLIT TTGGCCTGTACTGCACA\n(ImmE1) KNSEGYYF CTTATATATATATACCT\n(Microcin- LVSDKML TATAACAAAAAACAGC\nE1 YAIVISTIL GAAGGGTATTATTTCCT\nimmunity CPYSKYAI TGTGTCAGATAAGATG\nmodulator) EYIAFNFIK CTATATGCAATAGTGAT\nKDFFERRK AAGCACTATTCTATGTC\nNLNNAPVA CATATTCAAAATATGCT\nKLNLFMLY ATTGAATACATAGCTTT\nNLLCLVLA TAACTTCATAAAGAAA\nIPFGLLGLF GATTTTTTCGAAAGAAG\nISIKNN AAAAAACCTAAATAAC\nGCCCCCGTAGCAAAATT\nAAACCTATTTATGCTAT\nATAATCTACTTTGTTTG\nGTCCTAGCAATCCCATT\nTGGATTGCTAGGACTTT\nTTATATCAATAAAGAAT\nAATTAA\n458 Cloacin MGLKLHIH Escherichia 459 ATGGGGCTTAAATTAC\nimmunity WFDKKTEE coli ATATTCATTGGTTTGAT\nmodulator FKGGEYSK AAGAAAACCGAAGAGT\nDFGDDGSV TTAAAGGCGGTGAATA\nIESLGMPL CTCAAAAGACTTCGGT\nKDNINNG GATGATGGTTCTGTCAT\nWFDVEKP TGAAAGTCTGGGGATG\nWVSILQPH CCTTTAAAGGATAATAT\nFKNVIDISK TAATAATGGTTGGTTTG\nFDYFVSFV ATGTTGAAAAACCATG\nYRDGNW GGTTTCGATATTACAGC\nCACACTTTAAAAATGTA\nATCGATATTAGTAAATT\nTGATTACTTTGTATCCT\nTTGTTTACCGGGATGGT\nAACTGGTAA\n460 Colicin-E2 MELKHSIS Escherichia 461 ATGGAACTGAAACATA\nimmunity DYTEAEFL coli GTATTAGTGATTATACC\nmodulator EFVKKICR GAGGCTGAATTTCTGG\n(ImmE2) AEGATEED AGTTTGTAAAAAAAAT\n(Microcin- DNKLVREF ATGTAGAGCTGAAGGT\nE2 ERLTEHPD GCTACTGAAGAGGATG\nimmunity GSDLIYYP ACAATAAATTAGTGAG\nmodulator) RDDREDSP AGAGTTTGAGCGATTA\nEGIVKEIKE ACTGAGCACCCAGATG\nWRAANGK GTTCAGATCTGATTTAT\nSGFKQG TATCCTCGCGATGACAG\nGGAAGATAGTCCTGAA\nGGGATTGTCAAGGAAA\nTTAAAGAATGGCGAGC\nTGCTAACGGTAAGTCA\nGGATTTAAACAGGGCT\nGA\n462 Colicin-A MMNEHSID Citrobacter 463 ATGATGAATGAACACT\nimmunity TDNRKAN freundii CAATAGATACGGACAA\nmodulator NALYLFIII CAGAAAGGCCAATAAC\n(Microcin- GLIPLLCIF GCATTGTATTTATTTAT\nA immunity VVYYKTPD AATAATCGGATTAATAC\nmodulator) ALLLRKIA CATTATTGTGCATTTTT\nTSTENLPSI GTTGTTTACTACAAAAC\nTSSYNPLM GCCAGACGCTTTACTTT\nTKVMDIYC TACGTAAAATTGCTACA\nKTAPFLALI AGCACTGAGAATCTCCC\nLYILTFKIR GTCAATAACATCCTCCT\nKLINNTDR ACAACCCATTAATGACA\nNTVLRSCL AAGGTTATGGATATTTA\nLSPLVYAA TTGTAAAACAGCGCCTT\nIVYLFCFR TCCTTGCCTTAATACTA\nNFELTTAG TACATCCTAACCTTTAA\nRPVRLMAT AATCAGAAAATTAATC\nNDATLLLF AACAACACCGACAGGA\nYIGLYSIIFF ACACTGTACTTAGATCT\nTTYITLFTP TGTTTATTAAGTCCATT\nVTAFKLLK GGTCTATGCAGCAATTG\nKRQ TTTATCTATTCTGCTTC\nCGAAATTTTGAGTTAAC\nAACAGCCGGAAGGCCT\nGTCAGATTAATGGCCA\nCCAATGACGCAACACT\nATTGTTATTTTATATTG\nGTCTGTACTCAATAATT\nTTCTTTACAACCTATAT\nCACGCTATTCACACCAG\nTCACTGCATTTAAATTA\nTTAAAAAAAAGGCAGT\nAA\n464 Colicin-Ia MNRKYYF Escherichia 465 ATGAACAGAAAATATT\nimmunity NNMWWG coli ATTTTAATAATATGTGG\nmodulator WVTGGYM TGGGGATGGGTGACGG\nLYMSWDY GGGGATATATGCTGTA\nEFKYRLLF TATGTCATGGGATTATG\nWCISLCGM AGTTTAAATACAGATTA\nVLYPVAK CTGTTCTGGTGTATTTC\nWYIEDTAL TCTCTGCGGAATGGTTT\nKFTRPDFW TGTATCCGGTTGCAAAA\nNSGFFADT TGGTATATTGAAGATAC\nPGKMGLLA AGCTCTAAAATTTACCC\nVYTGTVFI GGCCTGATTTCTGGAAC\nLSLPLSMIY AGCGGTTTTTTTGCTGA\nILSVIIKRLS TACACCTGGAAAAATG\nVR GGGTTGCTTGCGGTTTA\nTACGGGTACTGTTTTCA\nTATTATCTCTTCCGTTA\nAGTATGATATATATTCT\nTTCTGTTATTATAAAAA\nGGCTGTCTGTAAGATAG\n466 Colicin-Ib MKLDISVK Escherichia 467 ATGAAACTGGATATATC\nimmunity YLLKSLIPI coli TGTAAAGTATTTACTGA\nmodulator LIILTVFYL AAAGCCTGATACCAAT\nGWKDNQE CCTCATTATTCTTACAG\nNARMFYAF TTTTTTATCTGGGATGG\nIGCIISAITF AAAGATAACCAGGAAA\nPFSMRIIQK ATGCAAGAATGTTTTAT\nMVIRFTGK GCGTTCATCGGATGCAT\nEFWQKDFF TATCAGTGCCATTACTT\nTNPVGGSL TTCCTTTTTCAATGAGG\nTAIFELFCF ATAATACAGAAAATGG\nVISVPVVAI TAATAAGGTTTACAGG\nYLIFILCKA GAAAGAATTCTGGCAA\nLSGK AAAGACTTCTTTACAAA\nTCCAGTTGGCGGAAGC\nTTAACTGCAATATTTGA\nATTATTCTGTTTCGTTA\nTATCAGTTCCTGTGGTT\nGCCATTTACTTAATTTT\nTATACTCTGCAAAGCCC\nTTTCAGGAAAATGA\n468 Colicin-N MHNTLLEK Escherichia 469 ATGCACAATACACTCCT\nimmunity IIAYLSLPG coli CGAAAAAATCATCGCA\nmodulator FHSLNNPP TACCTATCCCTACCAGG\n(Microcin- LSEAFNLY ATTTCATTCATTAAACA\nN immunity VHTAPLAA ACCCGCCCCTAAGCGA\nmodulator) TSLFIFTHK AGCATTCAATCTCTATG\nELELKPKS TTCATACAGCCCCTTTA\nSPLRALKIL GCTGCAACCAGCTTATT\nTPFTILYIS CATATTCACACACAAAG\nMIYCFLLT AATTAGAGTTAAAACC\nDTELTLSS AAAGTCGTCACCTCTGC\nKTFVLIVK GGGCACTAAAGATATT\nKRSVFVFF AACTCCTTTCACTATTC\nLYNTIYWD TTTATATATCCATGATA\nIYIHIFVLL TACTGTTTCTTGCTAAC\nVPYRNI TGACACAGAACTAACC\nTTGTCATCAAAAACATT\nTGTATTAATAGTCAAAA\nAACGATCTGTTTTTGTC\nTTTTTTCTATATAACAC\nTATATATTGGGATATAT\nATATTCACATATTTGTA\nCTTTTGGTTCCTTATAG\nGAACATATAA\n470 Colicin-E8 MELKNSIS Escherichia 471 ATGGAACTGAAAAACA\nimmunity DYTETEFK coli GCATTAGTGATTACACT\nmodulator KIIEDIINCE GAAACTGAATTCAAAA\n(ImmE8) GDEKKQD AAATTATTGAAGACATC\n(Microcin- DNLEHFIS ATCAATTGTGAAGGTG\nE8 VTEHPSGS ATGAAAAAAAACAGGA\nimmunity DLIYYPEG TGATAACCTCGAGCATT\nmodulator) NNDGSPEA TTATAAGTGTTACTGAG\nVIKEIKEW CATCCTAGTGGTTCTGA\nRAANGKSG TCTGATTTATTACCCAG\nFKQG AAGGTAATAATGATGG\nTAGCCCTGAAGCTGTTA\nTTAAAGAGATTAAAGA\nATGGCGAGCTGCTAAC\nGGTAAGTCAGGATTTA\nAACAGGGCTGA\n472 Lactococcin-A MKKKQIEF Lactococcus 473 ATGAAAAAAAAACAAA\nimmunity ENELRSML lactis TAGAATTTGAAAACGA\nmodulator ATALEKDI subsp. GCTAAGAAGTATGTTG\nSQEERNAL lactis GCTACCGCCCTTGAAAA\nNIAEKALD (Streptococcus AGACATTAGTCAAGAG\nNSEYLPKII lactis) GAAAGAAATGCTCTGA\nLNLRKALT ATATTGCAGAAAAGGC\nPLAINRTL GCTTGACAATTCTGAAT\nNHDLSELY ATTTACCAAAAATTATT\nKFITSSKAS TTAAACCTCAGAAAAG\nNKNLGGG CCCTAACTCCATTAGCT\nLIMSWGRLF ATAAATCGAACACTTAA\nCCATGATTTATCTGAAC\nTGTATAAATTCATTACA\nAGTTCCAAAGCATCAA\nACAAAAATTTAGGTGG\nTGGTTTAATTATGTCGT\nGGGGACGACTATTCTAA\n474 Lactococcin-A MKKKQIEF Lactococcus 475 ATGAAAAAAAAACAAA\nimmunity ENELRSML lactis TAGAATTTGAAAACGA\nmodulator ATALEKDI subsp. GCTAAGAAGTATGTTG\nSQEERNAL cremoris GCTACCGCCCTTGAAAA\nNIAEKALD (Streptococcus AGACATTAGTCAAGAG\nNSEYLPKII cremoris) GAAAGAAATGCTCTGA\nLNLRKALT ATATTGCAGAAAAGGC\nPLAINRTL GCTTGACAATTCTGAAT\nNHDLSELY ATTTACCAAAAATTATT\nKFITSSKAS TTAAACCTCAGAAAAG\nNKNLGGG CCCTAACTCCATTAGCT\nLIMSWGRLF ATAAATCGAACACTTAA\nCCATGATTTATCTGAAC\nTGTATAAATTCATTACA\nAGTTCCAAAGCATCAA\nACAAAAATTTAGGTGG\nTGGTTTAATTATGTCGT\nGGGGACGACTATTCTAA\n476 Colicin-D MNKMAMI Escherichia 477 ATGATCGATTTGGCGA\nimmunity DLAKLFLA coli AATTATTTTTAGCTTCG\nmodulator SKITAIEFS AAAATTACAGTGATTG\n(Microcin- ERICVERR AGTTTTCAGAGCGAATT\nD immunity RLYGVKDL TGTGTTGAACGGAGAA\nmodulator) SPNILNCG GATTGTATGGTGTTAAG\nEELFMAAE GATTTGTCTCCGAATAT\nRFEPDADR ATTAAATTGTGGGGAA\nANYEIDDN GAGTTGTCTATGGCTGC\nGLKVEVRS TGAGCGATTTGAGCCT\nILEKFKL GATGCAGATAGGGCTA\nATTATGAAATTGATGAT\nAATGGACTTAAGGTCG\nAGGTCCGATCTATCTTG\nGAAAAACTTAAATCAT\nAA\n478 Colicin-E5 MKLSPKAA Escherichia 479 ATGAAGTTATCACCAA\nimmunity IEVCNEAA coli AAGCTGCAATAGAAGT\nmodulator KKGLWILG TTGTAATGAAGCAGCG\n(ImmE5) IDGGHWLN AAAAAAGGCTTATGGA\n(Microcin- PGFRIDSSA TTTTGGGCATTGATGGT\nE5 SWTYDMP GGGCATTGGCTGAATC\nimmunity EEYKSKIPE CTGGATTCAGGATAGA\nmodulator) NNRLAIENI TAGTTCAGCATCATGGA\nKDDIENGY CATATGATATGCCGGA\nTAFIITLKM GAATACAAATCAAAAA\nTCCCTGAAAATAATAG\nATTGGCTATTGAAAATA\nTTAAAGATGATATTGA\nGAATGGATACACTGCTT\nTCATTATCACGTTAA\n480 Colicin-E6 MGLKLHIN Escherichia 481 ATGGGGCTTAAATTAC\nimmunity WFDKRTEE coli ATATTAATTGGTTTGAT\nmodulator FKGGEYSK AAGACGACCGAGGAAT\n(ImmE6) DFGDDGSV TTAAAGGTGGTGAGTA\n(Microcin- IERLGMPF TTCAAAAGATTTTGGAG\nE6 KDNINNG ATGATGGCTCGGTCATT\nimmunity WFDVIAEW GAACGTCTTGGAATGC\nmodulator) VPLLQPYF CTTTAAAAGATAATATC\nNHQIDISD AATAATGGTTGGTTTGA\nNEYFVSFD TGTTATAGCTGAATGG\nYRDGDW GTACCTTTGCTACAACC\nATACTTTAATCATCAAA\nTTGATATTTCCGATAAT\nGAGTATTTTGTTTCGTT\nTGATTATCGTGATGGTG\nATTGGTGA\n482 Colicin-E8 MELKKSIG Escherichia 483 GTGGAGCTAAAGAAAA\nimmunity DYTETEFK coli GTATTGGTGATTACACT\nmodulator KIIENIINCE GAAACCGAATTCAAAA\nin ColE6 GDEKKQD AAATTATTGAAAACATC\n(E8Imm[E6]) DNLEHFIS ATCAATTGTGAAGGTG\nVTEHPSGS ATGAAAAAAAACAGGA\nDLIYYPEG TGATAACCTCGAGCATT\nNNDGSPEA TTATAAGTGTTACTGAG\nVIKEIKEW CATCCTAGTGGTTCTGA\nRAANGKSG TCTGATTTATTACCCAG\nFKQG AAGGTAATAATGATGG\nTAGCCCTGAAGCTGTTA\nTTAAAGAGATTAAAGA\nATGGCGAGCTGCTAAC\nGGTAAGTCAGGATTTA\nAACAGGGCTGA\n484 Colicin-E9 MELKHSIS Escherichia 485 ATGGAACTGAAGCATA\nimmunity DYTEAEFL coli GCATTAGTGATTATACA\nmodulator QLVTTICN GAAGCTGAATTTTTACA\n(ImmE9) ADTSSEEE ACTTGTAACAACAATTT\n(Microcin- LVKLVTHF GTAATGCGAACACTTCC\nE9 EEMTEHPS AGTGAAGAAGAACTGG\nimmunity GSDLIYYP TTAAATTGGTTACACAC\nmodulator) KEGDDDSP TTTGAGGAAATGACTG\nSGIVNTVK AGCACCCTAGTGGTAG\nQWRAANG TGATTTAATATATTACC\nKSGFKQG CAAAAGAAGGTGATGA\nTGACTCACCTTCAGGTA\nTTGTAAACACAGTAAA\nACAATGGCGAGCCGCT\nAACGGTAAGTCAGGAT\nTTAAACAGGGCTAA\n486 Colicin-M MLTLYGYI Escherichia 487 ATGAAAGTAATTAGCA\nimmunity RNVFLYR coli TGAAATTTATTTTTATT\nmodulator MNDRSCG TTAACGATTATTGCTCT\n(Microcin-M DFMKVISM TGCTGCTGTTTTTTTCT\nimmunity KFIFILTIIA GGTCTGAAGATAAAGG\nmodulator) LAAVFFWS TCCGGCATGCTATCAGG\nEDKGPACY TCAGCGATGAACAGGC\nQVSDEQAR CAGAACGTTTGTAAAA\nTFVKNDYL AATGATTACCTGCAAA\nQRMKRWD GAATGAAACGCTGGGA\nNDVQLLGT CAACGATGTACAACTTC\nEIPKITWEK TTGGTACAGAAATCCC\nIERSLTDVE GAAAATTACATGGGAA\nDEKTLLVP AAGATTGAGAGAAGTT\nFKAEGPDG TAACAGATGTTGAAGA\nKRMYYGM TGAAAAAACACTTCTTG\nYHCEEGY TCCCATTTAAAGCTGAA\nVEYAND GGCCCGGACGGTAAGA\nGAATGTATTATGGCATG\nTACCATTGTGAGGAGG\nGATATGTTGAATATGCG\nAATGACTAA\n488 Colicin-B MTSNKDK Escherichia 489 ATGACCAGCAATAAAG\nimmunity NKKANEIL coli ATAAGAACAAGAAAGC\nmodulator YAFSIIGIIP AAACGAAATATTATAT\n(Microcin- LMAILILRI GCATTTTCCATAATCGG\nB immunity NDPYSQVL GATTATTCCATTAATGG\nmodulator) YYLYNKV CTATATTAATACTTCGA\nAFLPSITSL ATAAATGATCCATATTC\nHDPVMTTL TCAAGTGCTGTACTACT\nMSNYNKT TATATAATAAGGTGGC\nAPVMGILV ATTTCTCCCTTCTATTA\nFLCTYKTR CATCATTGCATGATCCC\nEIIKPVTRK GTCATGACAACACTTAT\nLVVQSCFW GTCAAACTACAACAAG\nGPVFYAILI ACAGCGCCAGTTATGG\nYITLFYNLE GTATTCTCGTTTTTCTT\nLTTAGGFF TGCACATATAAGACAA\nKLLSHNVI GAGAAATCATAAAGCC\nTLFILYCSI AGTAACAAGAAAACTT\nYFTVLTMT GTTGTGCAATCCTGTTT\nYAILLMPL CTGGGGGCCCGTTTTTT\nLVIKYFKG ATGCCATTCTGATTTAT\nRQ ATCACACTGTTCTATAA\nTCTGGAACTAACAACA\nGCAGGTGGTTTTTTTAA\nATTATTATCTCATAATG\nTCATCACTCTGTTTATT\nTTATATTGCTCCATTTA\nCTTTACTGTTTTAACCA\nTGACATATGCGATTTTA\nCTGATGCCATTACTTGT\nCATTAAATATTTTAAAG\nGGAGGCAGTAA\n490 Colicin-V MDRKRTK Escherichia 491 ATGGATAGAAAAAGAA\nimmunity LELLFAFII coli CAAAATTAGAGTTGTTA\nmodulator NATAIYIAL TTTGCATTTATAATAAA\n(Microcin- AIYDCVFR TGCCACCGCAATATATA\nV immunity GKDFLSMH TTGCATTAGCTATATAT\nmodulator) TFCFSALM GATTGTGTTTTTAGAGG\nSAICYFVG AAAGGACTTTTTATCCA\nDNYYSISD TGCATACATTTTGCTTC\nKIKRRSYE TCTGCATTAATGTCTGC\nNSDSK AATATGTTACTTTGTTG\nGTGATAATTATTATTCA\nATATCCGATAAGATAA\nAAAGGAGATCATATGA\nGAACTCTGACTCTAAAT\nGA\n492 Colicin- MSLRYYIK Shigella 493 ATGAGTTTAAGATACTA\nE1* NILFGLYC sonnei CATAAAAAATATTTTGT\nimmunity ALIYIYLIT TTGGCCTATACTGCGCA\nmodulator KNNEGYYF CTTATATATATATACCT\n(ImmE1) LASDKMLY TATAACAAAAAACAAC\n(Microcin- AIVISTILCP GAAGGGTATTATTTCCT\nE1* YSKYAIEHI AGCGTCAGATAAGATG\nimmunity FFKFIKKDF CTATACGCAATAGTGAT\nmodulator) FRKRKNLN AAGCACTATTCTATGCC\nKCPRGKIK CATATTCAAAATATGCT\nPYLCVYNL ATTGAACACATATTTTT\nLCLVLAIPF TAAGTTCATAAAGAAA\nGLLGLVYI GATTTTTTCAGAAAAAG\nNKE AAAAAACCTAAATAAA\nTGCCCCCGTGGCAAAA\nTTAAACCGTATTTATGC\nGTATACAATCTACTTTG\nTTTGGTCCTAGCAATCC\nCATTTGGATTGCTAGGA\nCTTGTTTATATCAATAA\nAGAATAA\n494 Colicin-E1 MSLRYYIK Escherichia 495 ATGAGCTTAAGATACTA\nimmunity NILFGLYC coli CATAAAAAATATTTTAT\nmodulator TLIYIYLIT TTGGCCTGTACTGCACA\n(ImmE1) KNSEEYYF CTTATATATATATACCT\n(Microcin- LVTDKML TATAACAAAAAACAGC\nE1 YAIVISTIL GAAGAGTATTATTTCCT\nimmunity CPYSKYAI TGTGACAGATAAGATG\nmodulator) EHIAFNFIK CTATATGCAATAGTGAT\nKHFFERRK AAGCACTATTCTATGTC\nNLNNAPVA CATATTCAAAATATGCT\nKLNLFMLY ATTGAACACATAGCTTT\nNLLCLVLA TAACTTCATAAAGAAAC\nIPFGLLGLF ATTTTTTCGAAAGAAGA\nISIKNN AAAAACCTAAATAACG\nCCCCCGTAGCAAAATTA\nAACCTATTTATGCTATA\nTAATCTACTTTGTTTGG\nTCCTAGCAATCCCATTT\nGGATTGCTAGGACTTTT\nTATATCAATAAAGAATA\nATTAA\n496 Probable MRKNNILL Leuconostoc 497 TTGAGAAAAAATAACA\nleucocin-A DDAKIYTN gelidum TTTTATTGGACGATGCT\nimmunity KLYLLLID AAAATATACACGAACA\nmodulator RKDDAGY AACTCTATTTGCTATTA\nGDICDVLF ATCGATAGAAAAGATG\nQVSKKLDS ACGCTGGGTATGGAGA\nTKNVEALI TATTTGTGATGTTTTGT\nNRLVNYIRI TTCAGGTATCCAAAAA\nTASTNRIKF ATTAGATAGCACAAAA\nSKDEEAVII AATGTAGAAGCATTGA\nELGVIGQK TTAACCGATTGGTCAAT\nAGLNGQY TATATACGAATTACCGC\nMADFSDKS TTCAACAAACAGAATTA\nQFYSIFER AGTTTTCAAAAGATGA\nAGAGGCTGTAATTATA\nGAACTTGGTGTAATTG\nGTCAGAAGGCTGGATT\nAAACGGCCAATACATG\nGCTGATTTTTCTGACAA\nATCTCAGTTTTATAGTA\nTCTTTGAAAGATAA\n498 Lactococcin-B MKKKVDT Lactococcus 499 ATGAAAAAAAAAGTTG\nimmunity EKQITSWA lactis ATACAGAAAAACAAAT\nmodulator SDLASKNE subsp. TACTTCTTGGGCATCTG\nTKVQEKLI cremoris ACTTAGCTTCCAAAAAT\nLSSYIQDIE (Streptococcus GAAACAAAGGTTCAAG\nNHVYFPKA cremoris) AAAAATTAATACTGTCT\nMISLEKKL TCTTATATTCAGGACAT\nRDQNNICA CGAAAACCATGTTTACT\nLSKEVNQF TTCCAAAAGCAATGATT\nYFKVVEVN TCTTTAGAAAAAAAATT\nQRKSWMV ACGAGACCAAAATAAT\nGLIV ATTTGCGCTTTATCAAA\nAGAAGTCAATCAGTTTT\nATTTTAAAGTTGTTGAA\nGTAAATCAAAGAAAAT\nCCTGGATGGTAGGTTTG\nATAGTTTAA\n500 Pediocin MNKTKSE Pediococcus 501 ATGAATAAGACTAAGT\nPA-1 HIKQQALD acidilactici CGGAACATATTAAACA\nimmunity LFTRLQFLL ACAAGCTTTGGACTTAT\nmodulator QKHDTIEP TTACTAGGCTACAGTTT\n(Pediocin YQYVLDIL TTACTACAGAAGCACG\nACH ETGISKTK ATACTATCGAACCTTAC\nimmunity HNQQTPER CAGTACGTTTTAGATAT\nmodulator) QARVVYN TCTGGAGACTGGTATCA\nKIASQALV GTAAAACTAAACATAA\nDKLHFTAE CCAGCAAACGCCTGAA\nENKVLAAI CGACAAGCTCGTGTAG\nNELAHSQK TCTACAACAAGATTGCC\nGWGEFNM AGCCAAGCGTTAGTAG\nLDTTNTWP ATAAGTTACATTTTACT\nSQ GCCGAAGAAAACAAAG\nTTCTAGCAGCCATCAAT\nGAATTGGCGCATTCTCA\nAAAAGGGTGGGGCGAG\nTTTAACATGCTAGATAC\nTACCAATACGTGGCCTA\nGCCAATAG\n502 Putative MIKDEKIN Carnobacterium 503 ATGATAAAAGATGAAA\ncarnobacteriocin- KIYALVKS maltaromaticum AAATAAATAAAATCTAT\nBM1 ALDNTDV (Carnobacterium GCTTTAGTTAAGAGCGC\nimmunity KNDKKLSL piscicola) ACTTGATAATACGGAT\nmodulator LLMRIQET GTTAAGAATGATAAAA\nSINGELFY AACTTTCTTTACTTCTT\nDYKKELQP ATGAGAATACAAGAAA\nAISMYSIQ CATCAATTAATGGAGA\nHNFRVPDD ACTATTTTACGATTATA\nLVKLLALV AAAAAGAATTACAGCC\nQTPKAWS AGCTATTAGTATGTACT\nGF CTATTCAACATAACTTT\nCGGGTTCCTGACGATCT\nAGTAAAACTGTTAGCAT\nTAGTTCAAACACCTAAA\nGCTTGGTCAGGGTTTTAA\n504 Putative MDIKSQTL Carnobacterium 505 ATGGATATAAAGTCTCA\ncarnobacteriocin- YLNLSEAY maltaromaticum AACATTATATTTGAATC\nB2 KDPEVKAN (Carnobacterium TAAGCGAGGCATATAA\nimmunity EFLSKLVV piscicola) AGACCCTGAAGTAAAA\nmodulator QCAGKLTA GCTAATGAATTCTTATC\n(Carnocin- SNSENSYIE AAAATTAGTTGTACAAT\nCP52 VISLLSRGI GTGCTGGGAAATTAAC\nimmunity SSYYLSHK AGCTTCAAACAGTGAG\nmodulator) RIIPSSMLTI AACAGTTATATTGAAGT\nYTQIQKDI AATATCATTGCTATCTA\nKNGNIDTE GGGGTATTTCTAGTTAT\nKLRKYEIA TATTTATCCCATAAACG\nKGLMSVPY TATAATTCCTTCAAGTA\nIYF TGTTAACTATATATACT\nCAAATACAAAAGGATA\nTAAAAAACGGGAATAT\nTGACACCGAAAAATTA\nAGGAAATATGAGATAG\nCAAAAGGATTAATGTC\nCGTTCCTTATATATATT\nTCTAA\n506 Nisin MRRYLILI Lactococcus 507 ATGAGAAGATATTTAAT\nimmunity VALIGITGL lactis ACTTATTGTGGCCTTAA\nmodulator SGCYQTSH subsp. TAGGGATAACAGGTTT\nKKVRFDEG lactis ATCAGGGTGTTATCAA\nSYTNFIYD (Streptococcus ACAAGTCATAAAAAGG\nNKSYFVTD lactis) TGAGGTTTGACGAAGG\nKEIPQENV AAGTTATACTAATTTTA\nNNSKVKFY TTTATGATAATAAATCG\nKLLIVDMK TATTTCGTAACTGATAA\nSEKLLSSSN GGAGATTCCTCAGGAG\nKNSVTLVL AACGTTAACAATTCCAA\nNNIYEASD AGTAAAATTTTATAAGC\nKSLCMGIN TGTTGATTGTTGACATG\nDRYYKILP AAAAGTGAGAAACTTT\nESDKGAVK TATCAAGTAGCAACAA\nALRLQNFD AAATAGTGTGACTTTGG\nVTSDISDD TCTTAAATAATATTTAT\nNFVIDKND GAGGCTTCTGACAAGT\nSRKIDYMG CGCTATGTATGGGTATT\nNIYSISDTT AACGACAGATACTATA\nVSDEELGE AGATACTTCCAGAAAG\nYQDVLAE TGATAAGGGGGCGGTC\nVRVFDSVS AAAGCTTTGAGATTACA\nGKSIPRSE AAACTTTGATGTGACAA\nWGRIDKD GCGATATTTCTGATGAT\nGSNSKQSR AATTTTGTTATTGATAA\nTEWDYGEI AAATGATTCACGAAAA\nHSIRGKSLT ATTGACTATATGGGAA\nEAFAVEIN ATATTTACAGTATATCG\nDDFKLATK GACACCACCGTATCTGA\nVGN TGAAGAATTGGGAGAA\nTATCAGGATGTTTTAGC\nTGAAGTACGTGTGTTTG\nATTCAGTTAGTGGCAA\nAAGTATCCCGAGGTCT\nGAATGGGGGAGAATTG\nATAAGGATGGTTCAAA\nTTCCAAACAGAGTAGG\nACGGAATGGGATTATG\nGCGAAATCCATTCTATT\nAGAGGAAAATCTCTTA\nCTGAAGCATTTGCCGTT\nGAGATAAATGATGATT\nTTAAGCTTGCAACGAA\nGGTAGGAAACTAG\n508 Trifolitoxin MNDEICLT Rhizobium 509 ATGAATGATGAGATTT\nimmunity GGGRTTVT leguminosarum GCCTGACAGGTGGCGG\nmodulator RRGGVVY bv. ACGAACGACTGTCACG\nREGGPWSS trifolii CGGCGCGGCGGAGTCG\nTVISLLRHL TGTATCGCGAAGGCGG\nEASGFAEA CCCGTGGTCATCAACCG\nPSVVGTGF TCATTTCGCTCCTGCGG\nDERGRETL CATCTGGAAGCCTCTGG\nSFIEGEFVH CTTCGCTGAAGCTCCTT\nPGPWSEEA CCGTTGTCGGCACCGGT\nFPQFGMML TTCGATGAGCGCGGCC\nRRLHDATA GGGAGACATTATCGTTT\nSFKPPENS ATCGAGGGTGAGTTTG\nMWRDWFG TTCACCCAGGCCCTTGG\nRNLGEGQH TCGGAGGAGGCTTTTCC\nVIGHCDTG GCAATTTGGAATGATGT\nPWNIVCRS TGCGGCGACTGCACGA\nGLPVGLID TGCCACCGCCTCGTTCA\nWEVAGPV AACCTCCCGAAAACTC\nRADIELAQ GATGTGGCGCGATTGG\nACWLNAQ TTCGGGCGTAACCTCG\nLYDDDIAE GTGAGGGTCAACACGT\nRVGLGSVT AATAGGACACTGCGAC\nMRAHQVR ACAGGCCCATGGAACA\nLLLDGYGL TTGTTTGCCGGTCAGGA\nSRKQRGGF TTGCCTGTCGGGTTGAT\nVDKLITFA AGATTGGGAGGTGGCT\nVHDAAEQ GGGCCTGTCAGGGCGG\nAKEAAVTP ATATCGAATTGGCCCA\nESNDAEPL GGCTTGTTGGCTGAATG\nWAIAWRT CCCAGCTCTACGATGAC\nRSASWML GACATTGCGGAGAGGG\nHHRQTLEA TCGGATTAGGCTCTGTG\nALA ACCATGAGAGCGCATC\nAAGTTCGCCTGCTGCTT\nGACGGCTATGGTCTGTC\nTCGGAAGCAACGCGGC\nGGCTTCGTCGACAAGCT\nAATCACGTTCGCAGTTC\nACGATGCGGCCGAGCA\nGGCGAAAGAGGCGGCT\nGTCACGCCAGAGTCGA\nACGATGCGGAACCGCT\nATGGGCAATTGCCTGG\nCGCACTAGAAGTGCCT\nCCTGGATGCTCCATCAT\nCGGCAAACACTGGAAG\nCAGCGCTGGCATAG\n510 Antilisterial MNNIIPIMS Bacillus 511 ATGAATAACATAATCCC\nbacteriocin LLFKQLYS subtilis TATCATGTCTTTGCTGT\nsubtilosin RQGKKDAI (strain 168) TCAAACAGCTTTACAGC\nbiosynthesis RIAAGLVIL CGGCAAGGGAAAAAGG\nprotein AVFEIGLIR ACGCCATCCGCATTGCC\nAlbD QAGIDESV GCAGGCCTTGTCATTCT\nLRKTYIILA GGCCGTGTTTGAAATC\nLLLMNTY GGGCTGATCCGCCAGG\nMVFLSVTS CCGGCATTGATGAATC\nQWKESYM GGTGTTGCGCAAAACG\nKLSCLLPIS TATATCATACTCGCGCT\nSRSFWLAQ TCTTTTGATGAACACAT\nSVVLFVDT ATATGGTGTTTCTTTCC\nCLRRTLFFF GTGACATCACAATGGA\nILPLFLFGN AGGAATCTTATATGAA\nGTLSGAQT GCTGAGCTGCCTGCTGC\nLFWLGRFS CGATTTCTTCACGGAGC\nFFTVYSIIF TTTTGGCTCGCCCAGAG\nGVVLSNHF TGTCGTTTTGTTTGTCG\nVKKKNLM ATACCTGTTTGAGAAG\nFLLHAAIFA AACTTTATTCTTTTTTA\nCVCISAAL TTTTACCGCTGTTCTTA\nMPAATIPL TTTGGAAACGGAACGC\nCAVHILWA TGTCAGGGGCGCAAAC\nVVIDFPVFL ATTGTTTTGGCTCGGCA\nQAPPQQGK GGTTTTCGTTTTTTACC\nMHSFMRRS GTTTACTCCATTATTTT\nEFSFYKRE CGGAGTTGTGCTAAGC\nWNRFISSK AACCACTTCGTCAAAAA\nAMLLNYA GAAGAACTTGATGTTTC\nVMAVFSGF TGCTGCATGCGGCGAT\nFSFQMMNT ATTCGCCTGTGTATGTA\nGIFNQQVI TCAGCGCCGCTTTGATG\nYIVISALLL CCGGCCGCCACGATTCC\nICSPIALLY GCTTTGCGCGGTTCATA\nSIEKNDRM TCCTGTGGGCGGTGGT\nLLITLPIKR CATTGACTTTCCTGTCT\nKTMFWAK TTCTGCAGGCGCCTCCG\nYRFYSGLL CAGCAGGGCAAGATGC\nAGGFLLVV ATTCATTTATGCGGCGA\nMIVGFISGR TCTGAATTTTCGTTTTA\nSISVLTFLQ CAAAAGAGAATGGAAC\nCIELLLAG CGATTTATCTCTTCTAA\nAYIRLTAD AGCGATGCTGTTAAATT\nEKRPSFSW ACGCGGTAATGGCGGT\nQTEQQLWS ATTCAGCGGCTTCTTTT\nGFSKYRSY CGTTCCAGATGATGAA\nLFCLPLFLA CACCGGCATCTTCAATC\nILAGTAVS AGCAAGTGATTTATATC\nLAVIPIAGL GTGATTTCCGCGCTTTT\nVIVYYLQK GCTCATCTGCTCGCCGA\nQDGGFFDT TCGCCCTTTTGTATTCG\nSKRERLGS ATTGAAAAAAATGACC\nGGATGCTGCTCATCACG\nCTTCCGATCAAGCGAA\nAAACGATGTTTTGGGC\nGAAATATCGCTTTTATT\nCAGGCCTATTGGCAGG\nCGGATTTCTCCTTGTCG\nTGATGATTGTGGGTTTCA\n512 Putative MSILDIHD Bacillus 513 GCATTTTGGATATACAC\nABC VSVWYER subtilis GATGTATCCGTTTGGTA\ntransporter DNVILEQV (strain 168) TGAACGGGACAACGTC\nATP- DLHLEKGA ATCTTAGAGCACGTGG\nbinding VYGLLGV ACTTACACTTAGAAAAA\nprotein NGAGKTTL GGCGCCGTTTACGGATT\nAlbC INTLTGVN GCTTGGGGTAAACGGT\n(Antilisterial RNFSGRFT GCCGGCAAAACAACAC\nbacteriocin LCGIEAEA TGATCAATACGCTGACA\nsubtilosin GMPQKTSD GGAGTGAACCGCAATT\nbiosynthesis QLKTHRYF ACAGCGGGGGCTTTAC\nprotein AADYPLLF GCTGTGCGGCATTGAA\nAlbC) TEITAKDY GCTGAGGCCGGCATGC\nVSFVHSLY CGCAGAAAACATCAGA\nQKDFSEQQ TCAACTGAAGATTCACC\nFASLAEAF GTTACTTCGCCGCTGAT\nHFSKYINR TATCCGCTGCTGTTTAC\nRISELSLGN AGAAATTACGGCGAAG\nRQKVVLM GACTATGTGTCTTTCGT\nTGLLLRAP CCATTCGCTTTATCAAA\nLFILDEPLV AGGATTTTTCAGAGCG\nGLDVESIE ACAGTTTGCCAGTTTGG\nVFYQKMR CTGAGGCCTTTCATTTT\nEYCEAGGT TCAAAATACATCAACA\nILFSSHLLD GGAGAATCTCGGAGCT\nVVQRFCDY GTCCTTGGGGAACAGG\nAAILHNKQ CAAAAGGTTGTGTTGAT\nIQKVIPIGE GACAGGATTATTGCTGC\nETDLRREF GGGCTCCCCTGTTTATT\nFEVIGHE TTGGATGAGCCGCTCGT\nCGGTTTGGATGTGGAA\nTCAATAGAGGTCTTTTA\nTCAGAAAATGCGGGAG\nTACTGTGAGGAAGGCG\nGAACCATTTTGTTTTCT\nTCCCATCTGCTCGATGT\nCGTGCAGAGATTTTGTG\nATTTTGCGGCCATTCTG\nCACAACAAACAGATCC\nAAAAGGTCATTCCGATT\nGGGGAGGAGACCGATC\nTGCGGCGGGAATTTTTT\nGAGGTTATCGGCCATG\nAATAA\n514 Antilisterial MSPAQRRI Bacillus 515 TTGTCACCAGCACAAA\nbacteriocin LLYILSFIF subtilis GAAGAATTTTACTGTAT\nsubtilosin VIGAVVYF (strain 168) ATCCTTTCATTTATCTT\nbiosynthesis VKSDYLFT TGTCATCGGCGCAGTC\nprotein LIFIAIAILF GTCTATTTTGTCAAAAG\nAlbB GMRARKA CGATTATCTGTTTACGC\nDSR TGATTTTCATTGCCATT\nGCCATTCTGTTCGGGAT\nGCGCGCGCGGAAGGCT\nGACTCGCGATGA\n516 Colicin-E7 MELKNSIS Escherichia 517 ATGGAACTGAAAAATA\nimmunity DYTEAEFV coli GTATTAGTGATTACACA\nmodulator QLLKEIEK GAGGCTGAGTTTGTTCA\n(ImmE7) ENVAATD ACTTCTTAAGGAAATTG\n(Microcin- DVLDVLLE AAAAAGAGAATGTTGC\nE7 HFVKITEH TGCAACTGATGATGTGT\nimmunity PDGTDLIY TAGATGTGTTACTCGAA\nmodulator) YPSDNRDD CACTTTGTAAAAATTAC\nSPEGIVKEI TGAGCATCCAGATGGA\nKEWRAAN ACGGATCTGATTTATTA\nGKPGFKQG TCCTAGTGATAATAGA\nGACGATAGCCCCGAAG\nGGATTGTCAAGGAAAT\nTAAAGAATGGCGAGCT\nGCTAACGGTAAGCCAG\nGATTTAAACAGGGCTGA\n518 Pyocin-S1 MKSKISEY Pseudomonas 519 ATGAAGTCCAAGATTTC\nimmunity TEKEFLEF aeruginosa CGAATATACGGAAAAA\nmodulator VEDIYTNN GAGTTTCTTGAGTTTGT\nKKKFPTEE TGAAGACATATACACA\nSHIQAVLE AACAATAAGAAAAAGT\nFKKLTEHP TCCCTACCGAGGAGTCT\nSGSDLLYY CATATTCAAGCCGTGCT\nPNENREDS TGAATTTAAAAAACTAA\nPAGVVKEV CGGAACACCCAAGCGG\nKEWRASK CTCAGACCTTCTTTACT\nGLPGFKAG ACCCCAACGAAAATAG\nAGAAGATAGCCCAGCT\nGGAGTTGTAAAGGAAG\nTTAAAGAATGGCGTGC\nTTCCAAGGGGCTTCCTG\nGCTTTAAGGCCGGTTAG\n520 Pyocin-S2 MKSKISEY Pseudomonas 521 ATGAAGTCCAAGATTTC\nimmunity TEKEFLEF aeruginosa CGAATATACGGAAAAA\nmodulator VKDIYTNN (strain GAGTTTCTTGAGTTTGT\nKKKFPTEE ATCC TAAAGACATATACACA\nSHIQAVLE 15692/ AACAATAAGAAAAAGT\nFKKLTEHP PAO1/1C/ TCCCTACCGAGGAGTCT\nSGSDLLYY PRS 101/ CATATTCAAGCCGTGCT\nPNENREDS LMG TGAATTTAAAAAACTAA\nPAGVVKEV 12228) CGGAACACCCAAGCGG\nKEWRASK CTCAGACCTTCTTTACT\nGLPGFKAG ACCCCAACGAAAATAG\nAGAAGATAGCCCAGCT\nGGAGTTGTAAAGGAAG\nTTAAAGAATGGCGTGC\nTTCCAAGGGGCTTCCTG\nGCTTTAAGGCCGGTTAG\n522 Hiracin- MDFTKEEK Enterococcus 523 ATGGATTTTACTAAAGA\nJM79 LLNAISKV hirae AGAAAAACTTTTAAAT\nimmunity YNEATIDD GCAATTAGTAAAGTAT\nfactor YPDLKEKL ACAATGAAGCAACTAT\nFLYSKEISE AGATGACTATCCTGACT\nGKSVGEVS TAAAAGAAAAGCTCTTT\nMKLSSFLG CTTTATTCTAAAGAAAT\nRYILKHKF CAGTGAGGGAAAAAGT\nGLPKSLIEL GTTGGTGAAGTTAGTAT\nQEIVSKES GAAATTAAGTAGTTTTC\nQVYRGWA TTGGAAGATATATTTTA\nSIGIWS AAACATAAATTTGGATT\nACCTAAATCTTTAATAG\nAATTACAAGAAATTGTT\nAGTAAGGAATCTCAAG\nTATATAGAGGATGGGC\nTTCTATTGGTATTTGGA\nGTTAA\n524 Probable MKKKYRY Leuconostoc 525 TTGAAAAAAAAGTATC\nmesentericin- LEDSKNYT mesenteroides GGTATTTAGAAGATAG\nY105 STLYSLLV CAAAAATTACACTAGTA\nimmunity DNVDKPG CACTCTATTCTCTGTTA\nmodulator YSDICDVL GTTGATAATGTTGACAA\nLQVSKKLD ACCTGGATACTCAGATA\nNTQSVEAL TTTGCGATGTTTTGCTT\nINRLVNYIR CAAGTTTCTAAGAAGTT\nITASTYKIIF GGATAATACTCAAAGT\nSKKEEELII GTTGAAGCGCTAATTA\nKLGVIGQK ATCGATTGGTTAATTAT\nAGLNGQY ATTCGTATTACTGCTTC\nMADFSDKS AACATACAAAATTATTT\nQFYSVFDQ TTTCAAAAAAAGAAGA\nGGAATTGATTATAAAA\nCTTGGTGTTATTGGACA\nAAAAGCTGGACTTAAT\nGGTCAGTATATGGCTG\nATTTTTCAGACAAGTCT\nCAGTTTTACAGCGTTTT\nCGATCAGTAA\n526 Microcin- MSFLNFAF Escherichia 527 ATGAGTTTTCTTAATTT\n24 SPVFFSIMA coli TGCATTTTCTCCTGTAT\nimmunity CYFIVWRN TCTTCTCCATTATGGCG\nmodulator KRNEFVCN TGTTATTTCATTGTATG\nRLLSIIIISFL GAGAAATAAACGAAAC\nICFIYPWLN GAATTTGTCTGCAATAG\nYKIEVKYY ATTGCTATCAATTATAA\nIFEQFYLFC TAATATCTTTTTTGATA\nFLSSLVAV TGCTTCATATATCCATG\nVINLIVYFI GCTAAATTACAAAATC\nLYRRCI GAAGTTAAATATTATAT\nATTTGAACAGTTTTATC\nTTTTTTGTTTTTTATCGT\nCACTCGTGGCTGTTGTA\nATAAACCTAATTGTATA\nCTTTATATTATACAGGA\nGATGTATATGA\n528 Colicin-K MHLKYYL Escherichia 529 ATGCATTTAAAATACTA\nimmunity HNLPESLIP coli CCTACATAATTTACCTG\nmodulator WILILIFND AATCACTTATACCATGG\nNDNTPLLFI ATTCTTATTTTAATATT\nFISSIHVLL TAACGACAATGATAAC\nYPYSKLTIS ACTCCTTTGTTATTTAT\nRYIKENTK ATTTATATCATCAATAC\nLKKEPWYL ATGTATTGCTATATCCA\nCKLSALFY TACTCTAAATTAACCAT\nLLMAIPVG ATCTAGATATATCAAAG\nLPSFIYYTL AAAATACAAAGTTAAA\nKRN AAAAGAACCCTGGTAC\nTTATGCAAGTTATCTGC\nATTGTTTTATTTATTAA\nTGGCAATCCCAGTAGG\nATTGCCAAGTTTCATAT\nATTACACTCTAAAGAG\nAAATTAA\n530 Microcin MMIQSHPL Escherichia 531 ATGATGATACAATCTCA\nC7 self- LAAPLAVG coli TCCACTACTGGCCGCTC\nimmunity DTIGFFSSS CCCTGGCAGTAGGAGA\nmodulator APATVTAK TACAATTGGTTTCTTTT\nMccF NRFFRGVE CATCATCTGCTCCGGCA\nFLQRKGFK ACAGTTACTGCAAAAA\nLVSGKLTG ATCGTTTTTTTCGGGGA\nKTDFYRSG GTTGAGTTTCTTCAGAG\nTIKERAQE AAAGGGATTTAAGCTG\nFNELVYNP GTATCAGGGAAGCTTA\nDITCIMSTI CCGGTAAAACAGATTTT\nGGDNSNSL TATCGTTCAGGTACTAT\nLPFLDYDA TAAAGAAAGAGCTCAA\nIIANPKIIIG GAATTTAATGAGTTAGT\nYSDTTALL CTACAATCCTGATATTA\nAGIYAKTG CCTGTATAATGTCAACG\nLITFYGPAL ATCGGTGGAGATAACA\nIPSFGEHPP GTAATTCACTACTACCG\nLVDITYESF TTTCTGGACTATGATGC\nIKILTRKQS TATCATTGCAAACCCCA\nGIYTYTLP AAATTATCATAGGTTAC\nEKWSDESI TCAGATACAACTGCTTT\nNWNENKIL ATTAGCAGGAATATAT\nRPKKLYKN GCAAAAACAGGGTTAA\nNCAFYGSG TAACATTCTATGGACCA\nKVEGRVIG GCTCTTATTCCTTCGTT\nGNLNTLTG TGGTGAACATCCACCTC\nIWGSEWM TTGTGGATATAACATAT\nPEILNGDIL GAATCATTTATTAAAAT\nFIEDSRKSI ACTAACAAGAAAACAA\nATIERLFS TCAGGAATATATACCTA\nMLKLNRVF CACATTACCTGAAAAGT\nDKVSAIILG GGAGTGATGAGAGCAT\nKHELFDCA AAACTGGAATGAAAAC\nGSKRRPYE AAGATATTAAGGCCTA\nVLTEVLDG AGAAGCTATATAAAAA\nKQIPVLDG CAACTGTGCCTTTTATG\nFDCSHTHP GTTCCGGAAAAGTTGA\nMLTLPLGV GGGGCGTGTAATTGGA\nKLAIDFDN GGAAATCTAAATACTTT\nKNISITEQY GACAGGTATATGGGGG\nLSTEK AGTGAATGGATGCCTG\nAAATTCTTAATGGAGAT\nATATTGTTTATTGAGGA\nCAGTCGGAAAAGCATT\nGCAACAATTGAACGAT\nTATTCTCTATGCTAAAG\nCTTAATCGCGTGTTTGA\nTAAAGTTAGTGCAATA\nATACTCGGGAAACATG\nAGCTTTTTGATTGTGCA\nGGAAGTAAACGCAGAC\nCATATGAAGTATTAACA\nGAGGTATTAGATGGGA\nAACAGATTCCTGTACTG\nGATGGATTTGATTGTTC\nACATACACATCCAATGC\nTAACTCTTCCACTTGGT\nGTAAAATTAGCTATTGA\nCTTTGACAACAAAAATA\nTAT\n532 Sakacin-A MKADYKKI Lactobacillus 533 GGCAGATTATAAAAAA\nimmunity NSILTYTST sakei ATAAATTCAATACTAAC\nfactor ALKNPKIIK TTACACATCTACTGCTT\nDKDLVVLL TAAAAAACCCTAAAATT\nTIIQEEAKQ ATAAAAGATAAAGATT\nNRIFYDYK TAGTAGTCCTTCTAACT\nRKFRPAVT ATTATTCAAGAAGAAG\nRFTIDNNFE CCAAACAAAATAGAAT\nIPDCLVKL CTTTTATGATTATAAAA\nLSAVETPK GAAAATTTCGTCCAGC\nAWSGFS GGTTACTCGCTTTACAA\nTTGATAATAATTTTGAG\nATTCCTGATTGTTTGGT\nTAAACTACTGTCAGCTG\nTTGAAACACCTAAGGC\nGTGGTCTGGATTTAGTT\nAG\n534 Colicin-E5 MKLSPKAA Escherichia 535 TGAAGTTATCACCAAA\nimmunity IEVCNEAA coli AGCTGCAATAGAAGTT\nmodulator KKGLWILG TGTAATGAAGCAGCGA\nin ColE9 IDGGHWLN AAAAAGGCTTATGGAT\n(E5Imm[E9]) PGFRIDSSA TTTGGGCATTGATGGTG\nSWTYDMP GGCATTGGCTGAATCCT\nEEYKSKTP GGATTCAGGATAGATA\nENNRLAIE GTTCAGCATCATGGAC\nNIKDDIEN ATATGATATGCCGGAG\nGYTAFIITL GAATACAAATCAAAAA\nKM CCCCTGAAAATAATAG\nATTGGCTATTGAAAATA\nTTAAAGATGATATTGA\nGAATGGATACACTGCTT\nTCATTATCACGTTAAAG\nATGTAA\n536 Antilisterial MNNIFPIM Bacillus 537 TTGGGGAGGAGACCGA\nbacteriocin SLLFKQLY subtilis TCTGCGGCGGGAATTTT\nsubtilosin SRQGKKDA TTGAGGTTATCGGCCAT\nbiosynthesis IRIAAGLVI GAATAACATATTCCCCA\nprotein LAVFEIGLI TCATGTCGTTGCTGTTC\nAlbD RQAGIDES AAACAGCTGTACAGCC\nVLGKTYIIL GGCAAGGGAAAAAGGA\nALLLMNTY CGCTATCCGCATTGCTG\nMVFLSVTS CAGGGCTTGTGATTCTC\nQWKESYM GCCGTGTTTGAAATCG\nKLSCLLPIS GGCTGATCCGACAAGC\nSRSFWLAQ CGGCATTGACGAATCG\nSVVLFVDT GTGTTGGGAAAAACGT\nCLRRTLFFF ATATCATATTGGCGCTT\nILPLFLFGN CTCTTAATGAACACGTA\nGTLSGAQT TATGGTGTTTCTTTCCG\nLFWLGRFS TGACATCACAATGGAA\nFFTVYSILF GGAATCTTATATGAAG\nGVMLSNHF CTGAGCTGTCTGCTGCC\nVKKKNSM GATTTCATCACGGAGCT\nFLLHAAVF TTTGGCTCGCCCAGAGT\nAFVCLSAA GTCGTTCTGTTTGTCGA\nFMPAVTIP TACCTGTTTGAGAAGA\nLCAVHML ACGTTATTCTTTTTTAT\nWAVIIDFP TTTACCGCTGTTCTTAT\nVFLQAPPH TTGGAAACGGAACGCT\nQSKMHFF GTCAGGGGCGCAAACA\nMRRSEFSF TTGTTTTGGCTTGGCAG\nYKREWNR ATTTTCGTTTTTTACCG\nFISSKAMLL TTTACTCGATTCTATTC\nNYVVMAA GGAGTTATGCTAAGCA\nFSGFFSFQ ACCATTTCGTCAAAAAG\nMMNTGIFN AAGAACTCGATGTTTCT\nQQVIYIVIS GCTGCATGCGGCGGTA\nALLLICSPI TTCGCCTTTGTATGCCT\nALLYSIEK CAGTGCCGCTTTTATGC\nNDRMLLIT CGGCCGTCACGATCCC\nLPIKRRTM GCTATGCGCGGTTCACA\nFWAKYRF TGCTATGGGCGGTGAT\nYSGLLAGG CATTGACTTTCCGGTCT\nFLLVAIIVG TTCTGCAGGCGCCTCCG\nFISGRPISA CATCAGAGCAAGATGC\nLTFVQCME ATTTTTTTATGCGGCGA\nLLLAGAFIR TCTGAATTTTCGTTTTA\nLTADEKRP CAAAAGAGAATGGAAC\nSFGWQTEQ CGATTTATTTCTTCTAA\nQLWSGFSK AGCGATGCTGTTAAATT\nYRSYLFCL ACGTGGTGATGGCGGC\nPLFLATLA GTTCAGCGGATTCTTTT\nGTAVSLAV CGTTCCAGATGATGAA\nIPIAALIIVY CACTGGCATCTTCAATC\nYLQKQDG AGCAAGTGATTTATATT\nGFFDTSKR GTGATTTCCGCTCTATT\nERIGS GCTGATTTGCTCGCCGA\nTCGCCCTTTTGTACTCT\nATTGAAAAAAACGATC\nGCATGCTGCTCATCACG\nCTTCCAATTAAAAGAA\nGAACGATGTTTTGGGC\nGAAATATCGCTTTTATT\nCAG\n538 Microcin- MERKQKN Escherichia 539 ATGGAAAGAAAACAGA\nJ25 export SLFNYIYSL coli AAAACTCATTATTTAAT\nATP- MDVRGKF TATATTTATTCATTAAT\nbinding/permease LFFSMLFIT GGATGTAAGAGGTAAA\nprotein SLSSIIISISP TTTTTATTCTTTTCCAT\nMcjD LILAKITDL GTTATTCATTACATCAT\n(Microcin- LSGSLSNFS TATCATCGATAATCATA\nJ25 YEYLVLLA TCTATTTCACCATTGAT\nimmunity CLYMFCVI TCTTGCAAAGATTACAG\nmodulator) SNKASVFL ATTTACTGTCTGGCTCA\n(Microcin- FMILQSSLR TTGTCAAATTTTAGTTA\nJ25 INMQKKM TGAATATCTGGTTTTAC\nsecretion SLKYLREL TTGCCTGTTTATACATG\nATP- YNENITNL TTTTGCGTTATATCTAA\nbinding SKNNAGYT TAAAGCAAGTGTTTTTT\nprotein TQSLNQAS TATTTATGATACTGCAA\nMcjD) NDIYILVR AGTAGTCTACGTATTAA\nNVSQNILS CATGCAGAAAAAAATG\nPVIQLISTI TCGCTAAAGTATTTGAG\nVVVLSTKD AGAATTGTATAACGAA\nWFSAGVFF AATATAACTAACTTGAG\nLYILVFVIF TAAAAATAATGCTGGA\nNTRLTGSL TATACAACGCAAAGTCT\nASLRKHSM TAACCAGGCTTCAAATG\nDITLNSYSL ACATTTATATTCTTGTG\nLSDTVDN AGAAATGTTTCCCAGA\nMIAAKKNN ATATCCTGTCACCTGTT\nALRLISERY ATACAACTTATTTCCAC\nEDALTQEN TATTGTTGTTGTTTTAT\nNAQKKYW CTACGAAGGACTGGTTT\nLLSSKVLL TCTGCCGGTGTGTTTTT\nLNSLLAVIL TCTCTATATTCTGGTAT\nFGSVFIYNI TTGTAATTTTTAATACC\nLGVLNGV AGACTGACTGGCAGTTT\nVSIGHFIMI AGCGTCTCTCAGAAAA\nTSYIILLST CACAGCATGGATATCA\nPVENIGAL CTCTTAACTCTTATAGT\nLSEIRQSM CTGTTATCTGATACTGT\nSSLAGFIQR TGATAACATGATAGCA\nHAENKATS GCTAAAAAGAATAATG\nPSIPFLNME CATTAAGACTTATTTCT\nRKLNLSIRE GAACGTTATGAAGATG\nLSFSYSDD CTCTCACTCAGGAAAAC\nKKILNSVS AATGCTCAGAAAAAAT\nLDLFTGKM ACTGGTTACTCAGTTCT\nYSLTGPSG AAAGTTCTTTTATTGAA\nSGKSTLVK CTCTTTACTTGCTGTAA\nIISGYYKN TATTATTTGGTTCTGTA\nYFGDIYLN TTCATATATAATATTTT\nDISLRNISD AGGTGTGCTGAATGGT\nEDLNDAIY GTAGTTAGTATCGGCCA\nYLTQDDYI CTTCATTATGATTACAT\nFMDTLRFN CATATATCATTCTTCTT\nLRLANYDA TCAACGCCAGTGGAAA\nSENEIFKVL ATATAGGGGCATTGCT\nKLANLSVV AAGTGAGATCAGGCAG\nNNEPVSLD TCAATGTCTAGCCTGGC\nTHLINRGN AGGTTTTATTCAACGTC\nNYSGGQK ATGCCGAGAATAAAGC\nQRISLARLF CACATCTCCTTCAA\nLRKPAIIIID\nEATSALDY\nINESEILSSI\nRTHFPDALI\nINISHRINL\nLECSDCVY\nVLNEGNIV\nASGHFRDL\nMVSNEYIS\nGLASVTE\n540 Microcin MTLLSFGF Klebsiella 541 ATGACATTACTTTCATT\nE492 SPVFFSVM pneumoniae TGGATTTTCTCCTGTTT\nimmunity AFCIISRSK TCTTTTCAGTCATGGCG\nmodulator FYPQRTRN TTCTGTATCATTTCACG\nKVIVLILLT TAGTAAATTCTATCCGC\nFFICFLYPL AGAGAACGCGAAACAA\nTKVYLVGS AGTTATTGTTCTGATTT\nYGIFDKFY TACTAACTTTTTTTATT\nLFCFISTLI TGTTTTTTATATCCATT\nAIAINVVIL AACAAAAGTGTATCTG\nTINGAKNE GTGGGAAGTTACGGTA\nRN TATTTGACAAATTCTAC\nCTCTTTTGCTTTATTTC\nTACGTTAATTGCAATAG\nCAATTAACGTAGTGATA\nCTTACAATAAATGGAG\nCTAAGAATGAGAGAAA\nTTAG\n\nPoison-Antidote Systems”

It is possible to keep a specific microbial cells within a desired environment. This can include killing or stopping the growth of the microbial cells if they are not in the environment. These poison-antidote system, which are different from bacteriocins can be used to achieve such containment or for selective growth of microbial cell. U.S. Pat. describes several examples of poison antidote system. Nos. Nos. A poison-antidote combination may include a cytotoxic (poison), polypeptide and an antitoxin polypeptide (antidote), in one cell. A?poison nucleotide is the one used herein. A polynucleotide that encodes a poison polypeptide and an antidote Polynucleotide is a code for a poisonous polypeptide. Refers to a polynucleotide that encodes an antidote peptide.

“In some embodiments the poison polypeptide can be expressed constitutively while the antidote peptide can only be expressed under the desired conditions. Some embodiments only allow the poison polypeptide to be expressed in unfavorable conditions. The antidote polypeptide can only be expressed in desirable conditions. In some embodiments, the poison/antidote system can be configured to ensure that the microbial cells survive in desired environments but die under unfavorable conditions. In some embodiments, the poison antidote system can be configured to kill the microbial cells if they escape from an industrial environment. A poison antidote is also configured to ensure that the microbial cells survive when a vector (e.g. A plasmid encoding an antidote peptide dies when it is missing. Some embodiments encode the poison polypeptide in the host genome. The antidote peptide is encoded on a vector (such a plasmid, extrachromosomal array, episome, or minichromosome) and is only expressed when that vector is present in host cells. Some embodiments encode the poison polypeptide by a poison mononucleotide in a first vector. The antidote peptide is encoded on an antidote nucleotide in a second vector. As such, it is only expressed when that vector is present. In some embodiments, the presence or absence a recombination event (e.g. the integration of a polynucleotide sequencing encoding antidotepolynucleotide into a host genome) can determine whether the antidote oligotide is present. In some embodiments, expression of the antidote peptide is dependent on the presence of a vector or other recombination events. The poison and antidote peptide may be expressed constitutively. In some embodiments, where expression of antidotepolypeptide depends upon the presence of a vector, recombination events, or both, the expression of the poison and/or antidote can be conditional. This means that the poison polypeptide or antidote may only be expressed under conditions where the microbial cells are not desired and the antidote peptide only in those conditions.

“Exemplary microbial toxin polypeptide/antitoxin polypeptide pairs (also referred to as ?poison/antidote? Pairs that can be used in poison antidote system in conjunction with certain embodiments of this invention include, but not limited to, RelE/RelB and CcdB/CcdA. Many poison polypeptides such as RelE are extremely conserved in Gram-positive and Gram?negative bacteria and Archae. As such, they can be cytotoxic to a wide range of genetically modified and synthetic microbial cells. It is possible that an antidote peptide can inhibit the activity its poison partner in a wide range of host environments. This means that poison/antidote pairings such as those discussed herein can be used in a wide variety of genetically modified, naturally occurring and fully synthetic microbial cell types.

“A poison-antidote is different from a Bacteriocin system in that it provides an endogenous mechanism by which a microbe can kill or arrest itself. A bacteriocin provides an exogenous mechanism by which a microbe can kill or arrest another cell. A poison-antidote can only be used to kill or imprison the specific cell that produced the poison. However, some embodiments allow for a combination of a bacteriocin and poison-antidote systems. In some cases, a bacteriocin system described herein can be used to kill/arrest the growth of other cells in a culture. The poison-antidote may be used in conjunction with the bacteriocin-producing cell to stop it from growing in an unfriendly environment. The poison-antidote method can also be used to select bacteriocin-producing cells that have been genetically engineered so that they express an industrially useful molecule (an “industrially useful molecular”). In some cases, an antidote may be linked to the expression of an industrially-use molecule or bacteriocin. This can be done by either placing polynucleotides that encode the bacteriocin or the antidote or both under the control of one promoter. In some embodiments, the poison antidote system is also included in a bacteriocin-encoding microbial cell. The bacteriocin system can be used to regulate growth of microbial cells or other microbial cells in a specific environment. The poison-antidote is used for controlling microbial cells within that environment.

“Promoters”

“Promoters are well-known in the art. One or more genes can be driven by a promoter. A promoter can be used to drive the expression of a polynucleotide that encodes a desired gene product in some embodiments. A promoter can be used to drive expression of the bacteriocin polynucleotide in some embodiments. A promoter can drive expression of an immune modulator polynucleotide in some embodiments. A promoter can drive expression of a bacteriocin and an immunity modulator polynucleotide in some embodiments. A promoter can drive expression of a polynucleotide that encodes at least one of the following: a bacteriocin nucleotide, an immunity modulator, poison molecule or antidote molecular. Some promoters are able to drive transcription at all times (called “constitutive promotors?”). Some promoters are able to drive transcription in certain circumstances (conditional promoters). This could be due to the presence or absence a chemical compound, environmental condition, gene product, or stage of the cell’s cycle or other factors.

“The skilled artisan will know that depending on the expression activity desired, a promoter can be chosen and placed in cis along with the sequence to be expressed. Table 3.1-3.11 shows examples of promoters that have exemplary activities. A skilled artisan will recognize that not all promoters work with every transcriptional machine (e.g. RNA polymerases, general transcript factors, and other such things. As such, compatible species may not exist. While some promoters are described herein as compatible, it is possible that these promoters may also be used in other microorganisms.

The Biobricks foundation makes the promoters of Tables 3.1-3.11 publicly available. These promoters can be used in accordance to BioBrick, according to the Biobricks foundation Public Agreement (BPA), is encouraged.

“It is important to note that any of these?coding? “It should be appreciated that any of the?codings described herein (for instance, a bacteriocin, immunity, poison, antidote, antidote, product polynucleotide or bacteriocin) can generally be expressed under control by a desired promoter. One?coding may be used in some embodiments. A single promoter controls polynucleotide. In some cases, there may be more than one?coding. One promoter controls all polynucleotides. This could be two, three or four polynucleotides, five, six and seven polynucleotides, eight polynucleotides, nine polynucleotides, ten polynucleotides, or both. In some cases, this can be referred to as a “cocktail”. A single microorganism can produce a variety of bacteriocins. A promoter may control a bacteriocin-polynucleotide. A promoter may also control an immunity modulator in some embodiments. A promoter may control a polynucleotide that encodes a desired gene product in some embodiments. The same promoter may control both the bacteriocin and polynucleotides encoding the desired gene products in some embodiments. Different promoters may control the bacteriocin and polynucleotides encoding desired gene products in some embodiments. In some embodiments the promoter can control both the immunity modulator polynucleotide or the polynucleotide that encodes a desired gene product. In some cases, the immunity modulator and bacteriocin polynucleotide can be controlled by different promoters.

“Generally speaking, translation initiation of a transcript is controlled by sequences at or 5?” Which sequences are at or 5? End of the coding sequence for a transcript. A coding sequence may begin with a start codon that is designed to pair with an initiator transcript. Although Met (AUG is the most common start codon in naturally occurring translation systems), it will be obvious that an initiator transcript can be engineered so that it binds to any desired triplet. In certain instances, other triplets than AUG may also be used as start codons. Sequences located near the start codon may facilitate ribosomal assembly. For example, a Kozak sequence, (gcc)gccRccAUGG; SEQ ID NO. 542, where R is?A? Or?G? or?G?) A transcript may also include a?coding? in certain embodiments. A polynucleotide sequence such as a bacteriocin or immunity modulator sequence or polynucleotide that encodes a desired industrial product, includes a suitable start codon and translational sequence. Some embodiments may include multiple?coding. Each polynucleotide sequencing is placed in cis of a transcript. It contains a start codon and a translational initiation sequence. Some embodiments allow for multiple?coding. If two or more?coding sequences are placed in cis on a transcript then the two sequences are controlled by a single translation initiator sequence. They either provide a single protein that can function with both encoded cis polypeptides or provide a way to separate two cis polypeptides, such as a 2A sequence or similar. A translational intiator (tRNA) may be regulable, so that it regulates the initiation of translation for a bacteriocin or immunity modulator, poison, antidote, industrially useful molecule, and other bacteriocins.

“TABLE 3.1\nExemplary Metal-Sensitive Promoters\nSEQ\nID\nNO: Name Description Sequence\n544 BBa_I721001 Lead Promoter gaaaaccttgtcaatgaagagcgatctatg\n545 BBa_I731004 FecA promoter ttctcgttcgactcatagctgaacacaaca\n546 BBa_I760005 Cu-sensitive promoter atgacaaaattgtcat\n547 BBa_I765000 Fe promoter accaatgctgggaacggccagggcacctaa\n548 BBa_I765007 Fe and UV promoters ctgaaagcgcataccgctatggagggggtt\n549 BBa_J3902 PrFe (PI +?PII rus operon) tagatatgcctgaaagcgcataccgctatg”

“TABLE 3.2\nExemplary Cell Signaling-Responsive Promoters\nSEQ\nID\nNO: Name Description Sequence\n550 BBa_I1051 Lux cassette right promoter tgttatagtcgaatacctctggcggtgata\n551 BBa_I14015 P(Las) TetO ttttggtacactccctatcagtgatagaga\n552 BBa_I14016 P(Las) CIO ctttttggtacactacctctggcggtgata\n553 BBa_I14017 P(Rhl) tacgcaagaaaatggtttgttatagtcgaa\n554 BBa_I739105 Double Promoter (LuxR/HSL, cgtgcgtgttgataacaccgtgcgtgttga\npositive/cI, negative)\n555 BBa_I746104 P2 promoter in agr operon agattgtactaaatcgtataatgacagtga\nfrom S. aureus\n556 BBa_I751501 plux-cI hybrid promoter gtgttgatgcttttatcaccgccagtggta\n557 BBa_I751502 plux-lac hybrid promoter agtgtgtggaattgtgagcggataacaatt\n558 BBa_I761011 CinR, CinL and glucose acatcttaaaagttttagtatcatattcgt\ncontrolled promotor\n559 BBa_J06403 RhIR promoter repressible by tacgcaagaaaatggtttgttatagtcgaa\nCI\n560 BBa_J102001 Reverse Lux Promoter tcttgcgtaaacctgtacgatcctacaggt\n561 BBa_J64000 rhlI promoter atcctcctttagtcttccccctcatgtgtg\n562 BBa_J64010 lasI promoter taaaattatgaaatttgcataaattcttca\n563 BBa_J64067 LuxR +?3OC6HSL independent gtgttgactattttacctctggcggtgata\nR0065\n564 BBa_J64712 LasR/LasI Inducible & gaaatctggcagtttttggtacacgaaagc\nRHLR/RHLI repressible\nPromoter\n565 BBa_K091107 pLux/cI Hybrid Promoter acaccgtgcgtgttgatatagtcgaataaa\n566 BBa_K091117 pLas promoter aaaattatgaaatttgtataaattcttcag\n567 BBa_K091143 pLas/cI Hybrid Promoter ggttctttttggtacctctggcggtgataa\n568 BBa_K091146 pLas/Lux Hybrid Promoter tgtaggatcgtacaggtataaattcttcag\n569 BBa_K091156 pLux caagaaaatggtttgttatagtcgaataaa\n570 BBa_K091157 pLux/Las Hybrid Promoter ctatctcatttgctagtatagtcgaataaa\n571 BBa_K145150 Hybrid promoter: HSL-LuxR tagtttataatttaagtgttctttaatttc\nactivated, P22 C2 repressed\n572 BBa_K266000 PAI +?LasR ->?LuxI (AI) caccttcgggtgggcctttctgcgtttata\n573 BBa_K266005 PAI +?LasR ->?LasI & AI + aataactctgatagtgctagtgtagatctc\nLuxR –|LasI\n574 BBa_K266006 PAI +?LasR ->?LasI +?GFP & caccttcgggtgggcctttctgcgtttata\nAI +?LuxR –|LasI +?GFP\n575 BBa_K266007 Complex QS ->?LuxI & LasI caccttcgggtgggcctttctgcgtttata\ncircuit\n576 BBa_K658006 position 3 mutated promoter caagaaaatggtttgttatagtcgaataaa\nlux pR-3 (luxR & HSL\nregulated)\n577 BBa_K658007 position 5 mutated promoter caagaaaatggtttgttatagtcgaataaa\nlux pR-5 (luxR & HSL\nregulated)\n578 BBa_K658008 position 3&5 mutated caagaaaatggtttgttatagtcgaataaa\npromoter lux pR-3/5 (luxR &\nHSL regulated)\n579 BBa_R0061 Promoter (HSL-mediated luxR ttgacacctgtaggatcgtacaggtataat\nrepressor)\n580 BBa_R0062 Promoter (luxR & HSL caagaaaatggtttgttatagtcgaataaa\nregulated — lux pR)\n581 BBa_R0063 Promoter (luxR & HSL cacgcaaaacttgcgacaaacaataggtaa\nregulated – lux pL)\n582 BBa_R0071 Promoter (Rh1R & C4-HSL gttagctttcgaattggctaaaaagtgttc\nregulated)\n583 BBa_R0078 Promoter (cinR and HSL ccattctgctttccacgaacttgaaaacgc\nregulated)\n584 BBa_R0079 Promoter (LasR & PAI ggccgcgggttctttttggtacacgaaagc\nregulated)\n585 BBa_R1062 Promoter, Standard (luxR and aagaaaatggtttgttgatactcgaataaa\nHSL regulated — lux pR)”

“TABLE 3.3\nExemplary Constitutive E. coli ?70 Promoters\nSEQ\nID\nNO: Name Description Sequence\n586 BBa_I14018 P(Bla) gtttatacataggcgagtactctgttatgg\n587 BBa_I14033 P(Cat) agaggttccaactttcaccataatgaaaca\n588 BBa_I14034 P(Kat) taaacaactaacggacaattctacctaaca\n589 BBa_I732021 Template for Building Primer acatcaagccaaattaaacaggattaacac\nFamily Member\n590 BBa_I742126 Reverse lambda cI-regulated gaggtaaaatagtcaacacgcacggtgtta\npromoter\n591 BBa_J01006 Key Promoter absorbs 3 caggccggaataactccctataatgcgcca\n592 BBa_J23100 constitutive promoter family ggctagctcagtcctaggtacagtgctagc\nmember\n593 BBa_J23101 constitutive promoter family agctagctcagtcctaggtattatgctagc\nmember\n594 BBa_J23102 constitutive promoter family agctagctcagtcctaggtactgtgctagc\nmember\n595 BBa_J23103 constitutive promoter family agctagctcagtcctagggattatgctagc\nmember\n596 BBa_J23104 constitutive promoter family agctagctcagtcctaggtattgtgctagc\nmember\n597 BBa_J23105 constitutive promoter family ggctagctcagtcctaggtactatgctagc\nmember\n598 BBa_J23106 constitutive promoter family ggctagctcagtcctaggtatagtgctagc\nmember\n599 BBa_J23107 constitutive promoter family ggctagctcagccctaggtattatgctagc\nmember\n600 BBa_J23108 constitutive promoter family agctagctcagtcctaggtataatgctagc\nmember\n601 BBa_J23109 constitutive promoter family agctagctcagtcctagggactgtgctagc\nmember\n602 BBa_J23110 constitutive promoter family ggctagctcagtcctaggtacaatgctagc\nmember\n603 BBa_J23111 constitutive promoter family ggctagctcagtcctaggtatagtgctagc\nmember\n604 BBa_J23112 constitutive promoter family agctagctcagtcctagggattatgctagc\nmember\n605 BBa_J23113 constitutive promoter family ggctagctcagtcctagggattatgctagc\nmember\n606 BBa_J23114 constitutive promoter family ggctagctcagtcctaggtacaatgctagc\nmember\n607 BBa_J23115 constitutive promoter family agctagctcagcccttggtacaatgctagc\nmember\n608 BBa_J23116 constitutive promoter family agctagctcagtcctagggactatgctagc\nmember\n609 BBa_J23117 constitutive promoter family agctagctcagtcctagggattgtgctagc\nmember\n610 BBa_J23118 constitutive promoter family ggctagctcagtcctaggtattgtgctagc\nmember\n611 BBa_J23119 constitutive promoter family agctagctcagtcctaggtataatgctagc\nmember\n612 BBa_J23150 1bp mutant from J23107 ggctagctcagtcctaggtattatgctagc\n613 BBa_J23151 1bp mutant from J23114 ggctagctcagtcctaggtacaatgctagc\n614 BBa_J44002 pBAD reverse aaagtgtgacgccgtgcaaataatcaatgt\n615 BBa_J48104 NikR promoter, a protein of gacgaatacttaaaatcgtcatacttattt\nthe ribbon helix-helix family of\ntrancription factors that repress\nexpre\n616 BBa_J54200 lacq_Promoter aaacctttcgcggtatggcatgatagcgcc\n617 BBa_J56015 lacIQ – promoter sequence tgatagcgcccggaagagagtcaattcagg\n618 BBa_J64951 E. Coli CreABCD phosphate ttatttaccgtgacgaactaattgctcgtg\nsensing operon promoter\n619 BBa_K088007 GlnRS promoter catacgccgttatacgttgtttacgctttg\n620 BBa_K119000 Constitutive weak promoter of ttatgcttccggctcgtatgttgtgtggac\nlacZ\n621 BBa_K119001 Mutated LacZ promoter ttatgcttccggctcgtatggtgtgtggac\n622 BBa_K137029 constitutive promoter with atatatatatatatataatggaagcgtttt\n(TA)10 between ?10 and ?35\nelements\n623 BBa_K137030 constitutive promoter with atatatatatatatataatggaagcgtttt\n(TA)9 between ?10 and ?35\nelements\n624 BBa_K137031 constitutive promoter with ccccgaaagcttaagaatataattgtaagc\n(C)10 between ?10 and ?35\nelements\n625 BBa_K137032 constitutive promoter with ccccgaaagcttaagaatataattgtaagc\n(C)12 between ?10 and ?35\nelements\n626 BBa_K137085 optimized (TA) repeat tgacaatatatatatatatataatgctagc\nconstitutive promoter with 13\nbp between ?10 and ?35\nelements\n627 BBa_K137086 optimized (TA) repeat acaatatatatatatatatataatgctagc\nconstitutive promoter with 15\nbp between ?10 and ?35\nelements\n628 BBa_K137087 optimized (TA) repeat aatatatatatatatatatataatgctagc\nconstitutive promoter with 17\nbp between ?10 and ?35\nelements\n629 BBa_K137088 optimized (TA) repeat tatatatatatatatatatataatgctagc\nconstitutive promoter with 19\nbp between ?10 and ?35\nelements\n630 BBa_K137089 optimized (TA) repeat tatatatatatatatatatataatgctagc\nconstitutive promoter with 21\nbp between ?10 and ?35\nelements\n631 BBa_K137090 optimized (A) repeat aaaaaaaaaaaaaaaaaatataatgctagc\nconstitutive promoter with 17\nbp between ?10 and ?35\nelements\n632 BBa_K137091 optimized (A) repeat aaaaaaaaaaaaaaaaaatataatgctagc\nconstitutive promoter with 18\nbp between ?10 and ?35\nelements\n633 BBa_K256002 J23101:GFP caccttcgggtgggcctttctgcgtttata\n634 BBa_K256018 J23119:IFP caccttcgggtgggcctttctgcgtttata\n635 BBa_K256020 J23119:HO1 caccttcgggtgggcctttctgcgtttata\n636 BBa_K256033 Infrared signal reporter caccttcgggtgggcctttctgcgtttata\n(J23119:IFP:J23119:HO1)\n637 BBa_K292000 Double terminator + ggctagctcagtcctaggtacagtgctagc\nconstitutive promoter\n638 BBa_K292001 Double terminator + tgctagctactagagattaaagaggagaaa\nConstitutive promoter +?Strong\nRBS\n639 BBa_K418000 IPTG inducible Lac promoter ttgtgagcggataacaagatactgagcaca\ncassette\n640 BBa_K418002 IPTG inducible Lac promoter ttgtgagcggataacaagatactgagcaca\ncassette\n641 BBa_K418003 IPTG inducible Lac promoter ttgtgagcggataacaagatactgagcaca\ncassette\n642 BBa_M13101 M13K07 gene I promoter cctgtttttatgttattctctctgtaaagg\n643 BBa_M13102 M13K07 gene II promoter aaatatttgcttatacaatcttcctgtttt\n644 BBa_M13103 M13K07 gene III promoter gctgataaaccgatacaattaaaggctcct\n645 BBa_M13104 M13K07 gene IV promoter ctcttctcagcgtcttaatctaagctatcg\n646 BBa_M13105 M13K07 gene V promoter atgagccagttcttaaaatcgcataaggta\n647 BBa_M13106 M13K07 gene VI promoter ctattgattgtgacaaaataaacttattcc\n648 BBa_M13108 M13K07 gene VIII promoter gtttcgcgcttggtataatcgctgggggtc\n649 BBa_M13110 M13110 ctttgcttctgactataatagtcagggtaa\n650 BBa_M31519 Modified promoter sequence of aaaccgatacaattaaaggctcctgctagc\ng3.\n651 BBa_R1074 Constitutive Promoter I caccacactgatagtgctagtgtagatcac\n652 BBa_R1075 Constitutive Promoter II gccggaataactccctataatgcgccacca\n653 BBa_S03331 –Specify Parts List– ttgacaagcttttcctcagctccgtaaact”

“TABLE 3.4\nExemplary Constitutive E. coli ?s Promoters\nSEQ\nID\nNO: Name Description Sequence\n654 BBa_J45992 Full-length stationary phase ggtttcaaaattgtgatctatatttaacaa\nosmY promoter\n655 BBa_J45993 Minimal stationary phase osmY ggtttcaaaattgtgatctatatttaacaa\npromoter”

“TABLE 3.5\nExemplary Constitutive E. coli ?32 Promoters\nSEQ\nID\nNO: Name Description Sequence\n656 BBa_J45504 htpG Heat Shock Promoter tctattccaataaagaaatcttcctgcgtg”

“TABLE 3.6\nExemplary Constitutive B. subtilis ?A Promoters\nSEQ\nID\nNO: Name Description Sequence\n657 BBa_K143012 Promoter veg a constitutive aaaaatgggctcgtgttgtacaataaatgt\npromoter for B. subtilis\n658 BBa_K143013 Promoter 43 a constitutive aaaaaaagcgcgcgattatgtaaaatataa\npromoter for B. subtilis\n659 BBa_K780003 Strong constitutive promoter aattgcagtaggcatgacaaaatggactca\nfor Bacillus subtilis\n660 BBa_K823000 PliaG caagcttttcctttataatagaatgaatga\n661 BBa_K823002 PlepA tctaagctagtgtattttgcgtttaatagt\n662 BBa_K823003 Pveg aatgggctcgtgttgtacaataaatgtagt”

“TABLE 3.7\nExemplary Constitutive B. subtilis ?B Promoters\nSEQ\nID\nNO: Name Description Sequence\n663 BBa_K143010 Promoter ctc for B. subtilis atccttatcgttatgggtattgtttgtaat\n664 BBa_K143011 Promoter gsiB for B. subtilis taaaagaattgtgagcgggaatacaacaac\n665 BBa_K143013 Promoter 43 a constitutive aaaaaaagcgcgcgattatgtaaaatataa\npromoter for B. subtilis”

“TABLE 3.8\nExemplary Constitutive Promoters from miscellaneous prokaryotes\nSEQ\nID\nNO: Name Description Sequence\n666 a_K112706 Pspv2 from Salmonella tacaaaataattcccctgcaaacattatca\n667 BBa_K112707 Pspv from Salmonella tacaaaataattcccctgcaaacattatcg”

“TABLE 3.9\nExemplary Constitutive Promoters from bacteriophage T7\nSEQ\nID\nNO: Name Description Sequence\n668 BBa_I712074 T7 promoter (strong agggaatacaagctacttgttctttttgca\npromoter from T7\nbacteriophage)\n669 BBa_I719005 T7 Promoter taatacgactcactatagggaga\n670 BBa_J34814 T7 Promoter gaatttaatacgactcactatagggaga\n671 BBa_J64997 T7 consensus ?10 and rest taatacgactcactatagg\n672 BBa_K113010 overlapping T7 promoter gagtcgtattaatacgactcactatagggg\n673 BBa_K113011 more overlapping T7 agtgagtcgtactacgactcactatagggg\npromoter\n674 BBa_K113012 weaken overlapping T7 gagtcgtattaatacgactctctatagggg\npromoter\n675 BBa_R0085 T7 Consensus Promoter taatacgactcactatagggaga\nSequence\n676 BBa_R0180 T7 RNAP promoter ttatacgactcactatagggaga\n677 BBa_R0181 T7 RNAP promoter gaatacgactcactatagggaga\n678 BBa_R0182 T7 RNAP promoter taatacgtctcactatagggaga\n679 BBa_R0183 T7 RNAP promoter tcatacgactcactatagggaga\n680 BBa_Z0251 T7 strong promoter taatacgactcactatagggagaccacaac\n681 BBa_Z0252 T7 weak binding and taattgaactcactaaagggagaccacagc\nprocessivity\n682 BBa_Z0253 T7 weak binding promoter cgaagtaatacgactcactattagggaaga”

“TABLE 3.10\nExemplary Constitutive Promoters from yeast\nSEQ\nID\nNO: Name Description Sequence\n683 BBa_I766555 pCyc (Medium) Promoter acaaacacaaatacacacactaaattaata\n684 BBa_I766556 pAdh (Strong) Promoter ccaagcatacaatcaactatctcatataca\n685 BBa_I766557 pSte5 (Weak) Promoter gatacaggatacagcggaaacaacttttaa\n686 BBa_J63005 yeast ADH1 promoter tttcaagctataccaagcatacaatcaact\n687 BBa_K105027 cyc100 minimal promoter cctttgcagcataaattactatacttctat\n688 BBa_K105028 cyc70 minimal promoter cctttgcagcataaattactatacttctat\n689 BBa_K105029 cyc43 minimal promoter cctttgcagcataaattactatacttctat\n690 BBa_K105030 cyc28 minimal promoter cctttgcagcataaattactatacttctat\n691 BBa_K105031 cyc16 minimal promoter cctttgcagcataaattactatacttctat\n692 BBa_K122000 pPGK1 ttatctactttttacaacaaatataaaaca\n693 BBa_K124000 pCYC Yeast Promoter acaaacacaaatacacacactaaattaata\n694 BBa_K124002 Yeast GPD (TDH3) gtttcgaataaacacacataaacaaacaaa\nPromoter\n695 BBa_K319005 yeast mid-length ADH1 ccaagcatacaatcaactatctcatataca\npromoter\n696 BBa_M31201 Yeast CLB1 promoter accatcaaaggaagctttaatcttctcata\nregion, G2/M cell cycle\nspecific”

“TABLE 3.11\nExemplary Constitutive Promoters from miscellaneous\neukaryotes\nSEQ\nID\nNO: Name Description Sequence\n697 BBa_I712004 CMV promoter agaacccactgcttactggcttatcgaaat\n698 BBa_K076017 Ubc Promoter ggccgtttttggcttttttgttagacgaag”

The above-referenced promotors are only an example. A skilled artisan will quickly recognize that there are many variations of the promoters mentioned above, as well as many other promoters (including those isolated from naturally occurring organisms, variations thereof, or fully synthetic promoters), which can be easily used in accordance to some embodiments.

“Regulation of Gene Activity.”

“Gene activity can either be increased or decreased by regulating it. One embodiment of the gene product whose activity is controlled includes a bacteriocin or immunity modulator, poison molecule, antidote, molecule, and industrially useful molecules. One gene regulation system may regulate multiple gene products. Some embodiments regulate gene activity at the level gene expression. Some embodiments regulate gene activity at the transcriptional level. This could be done by activating or suppressing a promoter. Some embodiments regulate gene activity at the post-transcriptional levels, such as regulation of RNA stability. Some embodiments regulate gene activity at the translational level. This could be done by regulating the initiation of translation. Some embodiments regulate gene activity at the post-translational levels, such as regulation of polypeptide stability or post-translational modifications of the polypeptide or binding an inhibitor to that polypeptide.

“In some embodiments gene activity is increased. Some embodiments increase the activity of at least one of the following: bacteriocins, immunity modulators, industrially useful molecules, poison molecule or antidote molecules. The idea behind increasing gene activity is that it can be directly activated or decreased by an inhibitor of gene activation. Some embodiments activate gene activity by at least one of the following: Inducing promoter activation, inhibiting transcriptional repressors, increasing RNA stability or inducing a pre-transcriptional inhibitor (for instance, inducing a ribozyme (or antisense) oligonucleotide), initiating translation (for instance, via a regulatable transcriptomic (tRNA), or inducing a desired posttranslational modification or inhibiting an inhibitor (for ase that is directed to a protein coding gene) A compound that is present in the desired environment can induce a promoter. An iron-sensitive promoter can be used to induce transcription, as an example. A compound found in the desired culture medium may inhibit a transcriptional regulator. Tetracycline, for example, can be found in an environment to inhibit the transcriptional repressor and allow activity from the promoter tetO. A compound that is only found outside of the desired culture medium can induce transcription in some embodiments.

“In some embodiments, gene activation is decreased. The concept of decreasing gene activity is possible by either directly inducing gene activity or by decreasing activity of an activator. Some embodiments reduce gene activity, while maintaining some activity. Some embodiments completely inhibit gene activity. Some embodiments decrease gene activity by at least one of the following: activating a transcriptional regulator, inhibiting promoter activation, decreasing RNA stability or activating post-transcriptional inhibitors (e.g., expressing an antisense or ribozyme oligonucleotide), inactivating polypeptides (e.g., binding an inhibitor, or using a polypeptide specific protease), and failing to correctly localize polypeptides (e. Failure to secrete bacteriocin. Some embodiments reduce gene activity by removing a particular gene from a desired place, such as by using a FLP/FRT or crelox cassette to excise a gene or by the loss or degradation a plasmid. A gene product (e.g. A polypeptide or a product made by a gene products (e.g. The product of an enzyme reaction that inhibits further gene activity (e.g. a negative feedback loop).”

“Genetic Modifications of Microorganisms”

“Techniques for genetically altering microorganisms is well-known in the art. A microorganism can be genetically modified to contain a nucleic acid sequence that regulates the expression and encoding of at least one of the following: bacteriocins; immunity modulators; industrially useful molecules; poison molecules or antidote chemicals. Microorganisms can receive polynucleotides. They can either be stably integrated into their chromosomes or exist without the genome, such as in a plasmid or extrachromosomal array.

“Exemplary vectors to genetically modify microbial cells include viruses, plasmids and transposable element. It will also be apparent that complete microbial genomes containing desired sequences can be synthesized in a cell (see, for example, Gibson et al. Gibson et al. Science 329, 52-56 (2010). In some embodiments, a portion of a microbial gene (or a part thereof) is synthesized and then introduced into a microbial cells.

It is possible to genetically modify a microorganism to produce a desired type or spectrum of immunity modulator activity or bacteriocin. A cassette is available for inserting desired bacteriocin or immunity modulator polynucleotides in a polynucleotide sequencing. Examples of cassettes include, among others, a Cre/lox cassette and FLP/FRT cassette. Some embodiments of the cassette are placed on a DNA plasmid so that the desired combination of bacteriocin/or immunity modulator can be easily introduced to the microbial cells. Some embodiments position the cassette in the genome of a microbial cell so that the cassette with the desired combination of bacteriocin/or immunity modulator can be introduced to the desired place.

“Culture Media”

“Microbial culture environments may include a variety of media such as feedstocks. The application will determine the choice of the right culture medium. The conditions of a culture medium can include chemical composition as well as temperature, light levels, pH, CO2 and other factors.

“In some embodiments, a genetically engineered organism is added to a culture medium that contains other microorganisms as well as at least one feedstock. Some embodiments contain a compound that stimulates the activity of a bacteriocin or immunity modulator. In some cases, the culture medium contains a compound that inhibits or suppresses the expression or activity of a bacteriocin or immunity modulator. A compound that stimulates the activity is found outside the feedstock in some embodiments. A compound that inhibits the activity the immunity modulator may be present in some embodiments but not in the feedstock.

“The term “feedstock” is used in a broad sense to mean material that can be consumed, fermented, purified or modified by microbial organisms. “Feedstock” is used in this context in a broad meaning to include material that can be consumed or fermented, purified and modified by microbial organisms. As such, ?feedstock? It does not include food or other food products. A ‘feedstock’ is a term that can be used to refer to food products. is a type of culture medium. As such, ‘culture medium’ is used herein. It includes, but is not limited to, feedstock. As such, feedstocks are also considered when a?culture medium is mentioned. Feedstocks are also explicitly contemplated when a?culture medium” is mentioned.

“Genetically Engineered Microbial Cells.”

“In some instances, genetically modified cells for microbial growth are available. You can configure genetically modified microbial cell for many purposes. Some embodiments of microbial cells include genetic modifications that regulate at least one of the following: immunity modulators, bacteriocins or mollycose, poison molecules, antidote chemicals, and bacteriocins. Some embodiments include genetic modifications in microbial cells to regulate the expression bacteriocins. Some embodiments include genetic modifications in microbial cells to regulate expression of immunity modulators.

“In some embodiments, genetically modified microbial cell are modified to make a product. The product may be a gene product in some embodiments. For example, a polypeptide, or RNA. Polynucleotide?coding is a result. Sequence can be used to refer to sequences that encode either a polypeptide, or an RNA. Microbial cells can be programmed to produce one or more genes that help to synthesize a desired product. These include a carbohydrate or biofuel, lipid or small molecule. The activity of one or more genes from the microbial cells can be used to synthesize the product in some embodiments. Optionally, the product may also be synthesized by the activity of one or several gene products from one or more other microorganisms. Microbial cells can be programmed to remove or decontaminate one or more substances from a media, such as a feedstock, in some embodiments. One or more gene products from microbial cells can help to decontaminate the environment. Microbial cells can be programmed to search for materials, such as iron and rare earth metals.

“Controlling Microbial Cell Growth”

“In some instances, genetically modified microbial cell lines are used to regulate growth of other microbial species. In some cases, the microbial cell regulates the growth of other microbial cell strains or species, such as their own clones. In some instances, the microbial cell regulates the growth of microbial strains or species, such as invaders. A bacteriocin is secreted by microbial cells to regulate the growth of other microbial plants. Each microbial cell’s regulation can be affected by its expression (or absence thereof) of an immune modulator that has protective effects against particular bacteriocin.

“As used herein, a desired cell? The like and microbial cells with at least one characteristic are microbial cells that have at least one characteristic. In some embodiments, a desired cell is in an appropriate environment, for example its industrially-applicable feedstock. A desired cell can be a cell that has been positively selected for. This could include a cell with high levels of useful gene products or that has undergone particular recombination. A desired cell can be a cell that is capable of neutralizing contaminating cells such as pathogenic cells. A desired cell can be positively selected by the expression of an immunity modulator that corresponds to at least one bacteriocin. It is possible that a microbial cell which can neutralize other microbial cells without a similar neutralizing function would have a competitive advantage, but this theory is not binding. In some embodiments, the ability to neutralize other cells is what makes a desired cell desirable. A bacteriocin or a related immunity modulator can be used to positively select a desired cell.

“As used herein ?undesired cell? The microbial cells with at least one characteristic that makes survival, growth, and proliferation unfavorable are called the undesired cell. The undesired cell may be a invading microbial cells, such as a contaminating or invasive cell that has entered a culture medium. In some embodiments, an undesired cell has escaped from an appropriate culture medium, for example its industrially-applicable feedstock. An undesired cell may have lost a specific plasmid or failed to undergo a particular event of recombination. An undesired cell may not produce or produce the desired gene product in some embodiments. An undesired cell may be selected against in some embodiments. An undesired cell can be selected against by reducing its expression or activity of an immunity modifier that protects against abacin in the environment. An undesired cell can be selected against by reducing its expression or activity of an immune modulator that protects against the secretion of bacteriocin by the cell and its clones. An undesired cell can be selected against in some embodiments by decreasing the expression of bacteriocin in the cell, which puts it at a competitive disadvantage over other microbial cell types.

“FIG. “FIG. A first microbial cells is sometimes provided in some embodiments. Some embodiments provide a first microbial cells that secrete an active bacteriocin 100. Some embodiments do not want the first microbial cells 102. In some embodiments, for example, one or more of the earliest microbial cells being outside their industrial environment or lacking a desired environment condition for the first microbe can render the first microbe undesirable in a specific environment at a particular moment. If the first microbial cells are not desired, the immunity modulator, which corresponds to the bacteriocin, can be inactive. One or more of the immunity modulator promoters can be inactive, and an immunity modulator transcriptional regulator can be active. Post-transcriptional silencing can also occur (e.g. A ribozyme, antisense, or regulatable tRNA may occur. Post-transcriptional silencing (e.g. Site-specific protease or silencing posttranslational modification can occur, or a vector encoding a immunity modulator may be absent. If the first cell doesn’t have an active immune modulator, it is neutralized by the bacteriocin (142) produced by other cells in culture. A second microbial cell may grow 192 after the first cell has been neutralized.

“In some embodiments, it is desirable to have the first microbial cells 106. One or more of the desired microbial cells can be found within an industrial environment. The first microbial cells may also have undergone a recombination process or contain a specific vector. This makes the cell desirable in a particular environment. When a microbial cell produces an active immune modulator, it can do so when desired. In some cases, the first microbial cells can have one or more of the following: a constitutive inducer for immunity modulator polynucleotide; an activated promoter (but not necessarily constitutive); an inactive repressor for immunity modulator transcription; a regulatable transcript that is induced for production of the immune modulator; absence of post-translational or post-transcriptional silencing or a vector encoding it 136. The first microbial cells can survive in the absence of bacteriocin from the first microbial cells. A second microbial cells can either grow to 192 or be neutralized by the bacteriocin produced by the first microbial. This depends on whether or not the second microbial has 176 immunity modulator activity.

“In some embodiments, the second cell of the microbial family is desired 152. One or more desired recombination events may have occurred in the second cell. A desired vector can be present in the second cell. The second cell produces a product of greater value (e.g. A positive feedback loop, or the immune locus and desired product being under the exact same transcriptional control can make the second microbial cells desirable 162. The second microbial cells can be used to provide immunity modulator activity in order to protect against the bacteriocin or bacterocins produced by the first. In some cases, the second microbial cells can be designed so that the immunity modulator promoter (for example, a constitutive one) is active, an immunity modulator transcriptional regulator is inactive, an immunity modulator transcriptional suppressor is inactive, an immunity modulator transcriptional regulatory repressor is not inactive, a regulatable transcript (for the facilitation of the expression of an immunity modulator), and a lack post-translational silence (e.g. by site-specific protease of the immunity modulator or a vector encoding a immunity modulator may be present 182. In certain embodiments, the immunity modulator activity can be provided so that the second microbial cells can survive 192.

“In some embodiments, a secondary microbial cell may not be desired 156. One or more of the second cells could be an intruder (e.g. A contaminating cell, an unfavorable environmental condition for the second microbe (e.g. The presence or absence of an undesired condition or compound, the second microbial cells having produced product but not more (e.g. A negative feedback loop, or an immune modulator locus and desired products locus being under the exact same transcriptional control. Transcript levels are not desirable (e.g. The second microbial cells may become unattractive if they are unable to produce the desired product. In some embodiments, the immunity modulator activity of the second microbial cells can be insufficient or absent. 176 One or more of the immune modulator promoters can be inactive, and an immunity modulator transcriptional regulator can be active. Post-transcriptional silencing can also occur (e.g. A ribozyme, antisense oligonucleotide or ribozyme can be used to induce the immunity modulator. Post-transcriptional silencing can also occur (e.g. Site-specific protease or silencing posttranslational modification can occur, or an absence of a vector encoding the immunity modulator may occur 186. Some embodiments allow the first microbial cells to secrete bacteriocin activity 100. In some embodiments, the bacteriocin can kill the second microbial cells.

“One skilled in art will recognize that the steps, functions, and structures disclosed herein may be executed in a different order. The outlined functions or structures are just examples. Some functions and structure may be combined into smaller functions or structures. Other functions and/or structures can also be added without affecting the essence of the disclosed embodiments.

It can be used to control the growth of other microbial cell cultures in a culture for a wide range of genetically modified microbial organisms. A microbial cell can control the growth of other cells in the culture, according to some embodiments. Table 4 shows some examples of functions and configurations that a first microbial Cell can use to control the growth one or more other Microbial Cells according to certain embodiments.

“TABLE 4\nExemplary uses of bacteriocin systems in genetically modified\nmicrobial cells according to some embodiments herein\nExemplary Exemplary configurations\nFunction (according to some embodiments)\nBiological Immunity modulator activity\ncontainment: only in the desired culture\nmedium, but not outside and\nbacteriocin activity at least\noutside of the desired culture\nmedium; escape of the\nbacteriocin producing cell\noutside the desired culture\nenvironment results in cytotoxicity\nor growth inhibition of\nthe bacteriocin producing cell\nGenetic guard Bacteriocin constitutively produced;\ngenetic guard microbial organism does\nnot produce gene products for\nmodulating industrial process\nof interest; immunity\nmodulator constitutively produced\n(e.g under control of\nconstitutive promoter) and/or\ngenetic guard microbial\norganism is insensitive to\nthe bacteriocin (e.g. a S.\ncerevisiae genetic guard\nproducing bacteriocins that\ntarget E. coli)\nSelection of Desired recombination event\nrecombinants: causes an immunity\nmodulator to be restored in a\nbacteriocin-expressing host.\nAlternatively the immunity modulator\ncan be restored only\nafter the desired recombination event.\nVector stability: Immunity modulator (or at least\none gene essential for\nimmunity is encoded on a\nplasmid, and a corresponding\nbacteriocin locus is encoded on\nchromosome); clones that\nlose the desired plasmid lack\nimmunity and are neutralized\nby the bacteriocin\nMinimization of Immunity modulator activity\ngenetic drift dependent on production of\nindustrial product (e.g. immunity\nmodulator expression\ncontrolled by an operon, in which\na repressor is active in\nthe absence of industrial product,\nand inactive in the\npresence of industrial product);\nif a mutation causes the\nmicrobial organism’s production\nof industrial product to\nfall below a desired level or\ncease, the microbial organism\nceases to produce immunity\nmodulator, and is neutralized\nby the bacteriocin.\nSelection for Immunity modulator is\nmicrobes presenting co-expressed with the gene of\na high yield interest; microbial organisms\nof expression producing high levels of\nexpression (and/or gene product of interest can\nexpressing be selected by increasing\nclones) bacteriocin concentration;\nmicrobial organisms producing\nlow levels of gene product of\ninterest (e.g. Low industrial fitness? are neutralized\nDestruction during Desired microbial cells\nfermentation constitutively express at least one\nof contaminating type of bacteriocin; secreted\nmicrobes. bacteriocins neutralize\ninvading microbial cells\nDesired microbial cells express\nat least one type of\nbacteriocin when in the\ndesired environment (e.g.\nbacteriocin is under the control\nof an inducible promoter\nthat is activated by an\nintermediate of the fermentation\nprocess); secreted bacteriocins\nneutralize contaminating\ncells\nControl of the Immunity modulator activity\nratio of a is repressed by accumulated\nmicrobial flora, product made by a microbial\ncell; bacteriocins secreted by\nthe microbial cell (or other cells)\nneutralize the microbial\ncell”

“FIG. “FIG. 2” is a diagram showing a genetically engineered cell that controls the growth of at most one other microbial cells according to certain embodiments. A first microbial cells 200 may contain a bacteriocin and an immunity modulator polynucleotide. Optionally, the bacteriocin mononucleotide may be integrated into the cell’s DNA. The immunity modulator polynucleotide may be optionally integrated into a cell’s plasmid. Unwanted clones of the cell 210 can be created in some embodiments (a non-expressing clone). can be devoid of immunity modulator activity and can optionally lack bacteriocin activation. The non-expressing clone can be neutralized by the bacteriocin activity in the first microbial cells 200. An undesired cell clone 220 may lose a plasmid containing the immunity modulator polynucleotide. The undesired 220 clone can be neutralized by the bacteriocin activity in the first microbial cells 200. Some embodiments allow the microbial cells 230 to escape from the desired environment. This can cause the clone’s immunity modulator activity to be ineffective. The escaped cells 230 and/or the clones can neutralize the cell 230. The escaped cell 230 may also include a poison-antidote mechanism to kill the escaped cells upon their escape.

“FIG. “FIG. A first bacteriocin-polynucleotide can be found in the genetically engineered microbial cells 300. The second genetically engineered microbe 310 can contain a second bacteriocin oligotide. Each of the genetically engineered microbial cell (300 and 301) can contain a first immunity modulator and second immunity modulator polynucleotides. These polynucleotides encode resistance to the first bacteriocin. The second genetically engineered microbe 310 can become unwelcome and lose its first immunity modulator activity through any of the mechanisms described herein. It will then be controlled by the 300-generated first bacteriocin. The first bacteriocin 300 from the first genetically-engineered microbial cells and the second bacteriocin 310 from the second genetically-engineered microbial cells can neutralize an intruder cell.

“FIG. “FIG. The first genetically engineered cells 400 may contain at least one bacteriocin encoding first bacteriocin and at most one second bacteriocin encoding second bacteriocin. The first genetically engineered cells 400 can produce the first anti-invader cell bacteriocin 410. The second bacteriocin can be produced by the first genetically engineered cells 410 to neutralize a second intruder cell 420. Some embodiments allow the first invader to be of a different species or strain than the second invader. Some embodiments show that the first invader cells respond differently to different levels of bacteriocin activity from the second invader cells. Some embodiments show that the first invader cell occupies a different ecological niche to the second invader.

“FIG. “FIG.5” is a flow diagram that illustrates methods for controlling the growth at least one second microbial cells in culture, according to certain embodiments. This could include culturing a first-microbial cells in a culture medium that contains a second microbial. The conditions are such that the first microbial cells produce a bacteriocin sufficient to control growth of the second cell. Optionally, the first microbial cells can be maintained continuously for a time of 520. Some embodiments require that the first microbial cells are maintained continuously for at least three days. For example, 3, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 25, 35, 45, 55, 65, 70 or 75. 530. A change in culture media that indicates the presence or an increase in activity of a third microorganism can be detected. In response to the production of a second bacteriocin, the first microbial cells can be re-engineered. This will allow the third microbial cells to grow. In the conditions where the first microbial cells produce a bacteriocin sufficient to control growth of the second microbial cells, the re-engineered microbial cells can be grown in culture. You can continue to culture the re-engineered first microbial cell for up to 560 days. Some embodiments require that the culture of the re-engineered microorganism cell be maintained for at least three days. This could include at least 3, 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 25, 40, 45, 55, 65, 70 or 75.

“In certain embodiments, a first-microbial cell can control growth of a second microbe. A first microbial cells can control the growth of another microbial cells of the same strain. The cells of the strain may contain a bacteriocin and an immunity modulator polynucleotide. If the immunity modulator is expressed, it protects against the bacteriocin. If a strain clone loses the expression of the immunity modator, it can be neutralized with bacteriocin activity of the same strain. In some embodiments, immunity modulator polynucleotide may be in cis with the bacteriocin oligotide. This means that even if both the bacteriocin and immunity modulator polynucleotides are eliminated (e.g. If a plasmid has been lost or an FLP-FRT cassette has been excised, bacteriocin activity can still neutralize the cell. In certain embodiments, the immunity modulator protein is trans to the bacteriocin mononucleotide. If the immunity modulator activity of the microbial cells is not desired (e.g., if a virus is lost or if an environmental condition causes a decrease in immunity modulator activity), then it can be lost. The bacteriocin activity can be induced by both the microbial cells and other strain cells.

“In some embodiments, it is possible to control a ratio between two or more microbial strains or species. FIG. 3 illustrates an example control of ratios. FIG. 3 (see cells 300, 310). In some embodiments, a first microbial strain or species loses an immunity modulator activity via any of the mechanisms discussed herein when it is less desired than a bacteriocin-producing second strain or species, increasing the ratio of second strain or species to the first strain or species. A bacteriostatic or bacteriocin is used to control the ratio between a first and second strain/species. This allows for the control of growth to be easily reversed and/or minimizes the chance of either strain/species being eliminated. A first microbial species or strain can produce a first type of bacteriocin when it is stimulated by a promoter. This could be an intermediate or an industrially useful product. The levels of the bacteriocin will increase with increasing levels of the compound of concern. In some cases, the second microbial species or strain that produces the compound of interest (or catalyzes its production) may not be immune modulator active for the bacteriocin. The bacteriocin levels increase with increasing levels of the compound/substance of interest. This neutralizes the second strain, which lacks the appropriate immunity modulator or has insufficient immunity modulator activity to protect against the effects of the bacteriocin. The relative levels of the first and second strains increase. A first microbial strain may produce a first product with first bacteriocin activities, while a second strain will produce a second product with second bacteriocin activities. In some embodiments the first product and second product can be intermediates in the biosynthetic pathway. The first microbial strain may provide both a first and a second immunity modulator activitiy. In this case, the second immunity modulator activity could protect against the second Bacteriocin. However, the accumulation of the first product can negatively regulate the second immunity modulator activity (e.g. The presence of the first product can suppress expression of the second immune modulator, while the first immunity modulator activity may protect against the first. A second microbial strain may also be capable of providing a first and second immune modulator activity. However, the accumulation of the second product can negatively regulate the first immunity modulator activity (e.g. The presence of the second product can repress the expression of the first immune modulator. When a high level of the first product is present, the second immunity modulator of the first microbial species is activated and the microbial cells from the first strain are neutralized with the second bacteriocin. This increases the ratio of second strain to first strain and increases the relative amount second product to first. The second immunity modulator in a second microbial strain can be inactivated if a large amount of second product is accumulated. In this case, the microbial cells in the second strain will be neutralized by the first bacteriacin. This increases the ratio of first and second strains and decreases the amount of second product. Depending on the product level, the ratio between the first and second stain can be adjusted. In some embodiments, the ratios of first and second strains can be maintained in an equilibrium. In some embodiments, the equilibrium of ratios between the first and second products is maintained. In some embodiments, the second immunity modulator of the first microbial species responds to a first environment condition or compound. The ratio between the second and first microbial strains is controlled in the same way as above. In some embodiments the second microbial species’s first immunity modulator responds in a second environment condition or compound. The ratio between the first microbial strain and the second is controlled as above.

“In some embodiments it is desirable that a microbial cells be contained within a specific environment. For example, so that the first microbial cells can survive only in a certain culture medium like industrial feedstock. A microbial cell may contain a bacteriocin and an immunity modulator polynucleotide. In some cases, the immunity modulator corresponds with the bacteriocin. Some embodiments show that when the microbial cells are in a desired environment, they produce an active bacteriocin, and the corresponding immunity modulator. However, when the microbial cells escape the desired environment the active bacteriocin is produced but not the active immunity modulator. The microbial cells can be grown in any environment they choose, but are neutralized by their own bacteriocin if it escapes. In some embodiments, for example, the bacteriocin encoded in the bacteriocin mononucleotide can be expressed constitutively, while the immunity modulator can only be expressed when the microbial cells are in a desired environment. In some embodiments, for example, the bacteriocin encoded in the bacteriocin mononucleotide can be constitutively expressed while the immunity modulator can only be expressed when the microbial cells are in a desired environment. In some embodiments, the immune modulator’s transcriptional activator can only be present in the desired environment. In some embodiments, for example, the bacteriocin encoded in the bacteriocin mononucleotide as well as the immunity modulator are constitutively expressed. However, if the microbial cells escape, the immunity modulator can be deleted using the FLP-FRT method. It is possible that a genetic system to neutralize an escaped microbial cells is not used in the culture. This can lead to mutations that reduce or eliminate the functionality of the genetic system. If the microbial cell escapes, it is possible that the genetic system may cease to function. It is, however, appreciated that a bacteriocin/immunity modator system can be used both inside and outside of a culture to control growth of genetically engineered cells and/or neutralize invading microorganisms. Genetic drift can be minimized by using selective pressure according to some embodiments. This selective pressure can be used in accordance to some embodiments to help ensure that, if a microbial cell escapes from the desired environment, the bacteriocin/immunity module system will function to neutralize it. In some embodiments, a single genetically engineered circuit (e.g. a bacteriocin/immunity module system) can be used to both neutralize other microbial cultures in a desired environment and to further neutralize a microbial cell or its clones after they escape from that environment. According to some embodiments, it is possible to adjust the configurations of bacteriocins described herein so that the escaping microbial organism can be neutralized by its own bacteriacins and/or the bacteriocins from its direct or indirectly progeny and/or the bacteriocins in the cell of another escaped cell or its direct or indirect parent.

“Some embodiments allow a microbial cells to control growth in more than one way. A microbial cell may perform more than one function in some embodiments. In some cases, the microbial cells use the same bacteriocin/immunity moduleator pair for multiple functions. Some embodiments use a bacteriocin/immunity moduleator pair to perform a first function and another bacteriocin/immunity modator pair to perform a second function. In some embodiments, a microorganism can express a bacteriocin that limits the growth of non-expressed? bacteria. Clones that have lost immunity modulator activity can be used to provide containment in the desired environment. However, if the microbial cells are not in the desired environment, they may still express bacteriocin. FIG. illustrates a schematic illustration of these two forms of growth regulation. 2. In some embodiments, the first microbial cells can express a bacteriocin that inhibits the growth of the second microbial cells and can neutralize invading cells. FIG. 2 illustrates a schematic illustration of these two forms of growth regulation. 3. Some embodiments provide growth control using two or more bacteriocin immunity modulator pairs. In some cases, each type of growth control can be provided with a different pair of bacteriocin immunity modator pairs. A plasmid can have a first immune locus that encodes a desired product. A first bacteriocin will neutralize a clone that has lost the plasmid. The second immunity modulator polynucleotide, which corresponds to a second immune modulator, can be integrated into a microbial cell’s genome and can be silenced if the microbial cells escape from their desired environment. For example, the second immunity modator polypeptide can be found in an FLP-FRT cassette. This cassette is then excised after escape. The second bacteriocin can neutralize the microbial cells upon escape.

“It should be noted that some embodiments herein are compatible avec poison-antidote system. In some embodiments, a microbial cells, along with a bacteriocin or immunity modulator, also includes a poison-antidote device that kills or arrests the cell when it’s not in a desired environment.

“It is possible to control the growth and development of multiple types of microbial cell. An environment could contain, or potentially include, multiple types of undesirable microbial organisms. Different microbial organisms may be more susceptible to bacteriocins than others (for example, because they have different immunity modulators), so a mixture of two or more of bacteriocins is possible (e.g. A?cocktail? A?cocktail? (a mixture of bacteriocins and other microbial organisms) is useful in controlling their growth. One microbial cell may produce two or more bacteriocins. In some cases, this includes at least 2, 3, 5, 6, 7, 8, 9, 13, 14, 15, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or 50 different types of bacteriocins. In some embodiments, a mixture of two or more different bacteriocin-producing microbial cells are provided, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 different bacteriocin-producing microbial cells, including ranges between any two of the listed values. Optionally, one or more of the bacteriocin-producing microbial cells can produce two or more different bacteriocins.”

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