COMPOSITIONS AND METHODS OF USING INDUCIBLE SIGNALING FOR TUNABLE DYANMICS IN MICROBIAL COMMUNITIES

Abstract

Provided herein are compositions and methods for modulating induction of quorum sensing in bacterial cells. For example, provided herein is a method of inducing method of inducing quorum sensing, where the method includes: culturing a bacterial strain, wherein the bacterial strain comprises a first nucleic acid sequence encoding a first activator polypeptide, wherein expression of the first activator polypeptide produces a quorum sensing molecule precursor; a second nucleic acid sequence encoding a second activator polypeptide, wherein expression of the second activator polypeptide produces a quorum sensing; a third nucleic acid sequence encoding a third activator polypeptide that is capable of activating the quorum sensing system; a fourth nucleic acid sequence encoding a gene of interest, and contacting the bacterial strain with an inducer molecule; and converting the inducer molecule into a quorum sensing molecule, thereby allowing induction of quorum sensing.

Description

TECHNICAL FIELD

The present disclosure relates to compositions and methods for modulating induction of quorum sensing in bacterial cells.

BACKGROUND

Synthetic biology has the potential to revolutionize both healthcare and industry, with applications ranging from therapeutics and drug delivery, to bioproduction and bioremediation. These emerging applications have uncovered the need to engineer spatially extended complex multi-cellular populations, requiring new tools that can bridge the gap between single cell, population, and community level engineering. To achieve this, significant research efforts have been focused on engineering and characterizing a variety of cell-to-cell communication systems, with a particular focus on bacterial quorum sensing.

SUMMARY

This document provides compositions and methods for modulating induction of quorum sensing in bacterial cells. For example, provided herein are methods of inducing quorum sensing in a bacterial strain including nucleic acid sequences encoding activator polypeptides, bacterial strains including the nucleic acid sequences encoding the activator polypeptides, and methods of treating a patient using the bacterial strains described herein. The present disclosure is based on the discovery that an inducible quorum sensing system (iQS) enables a wide range of wide range of new functionality for synthetic circuits that rely on cell-to-cell communication systems for population and community level dynamics. In particular, inducibility broadly expands the functionality of quorum sensing based circuits by enabling switching between states of circuit quiescence, quorum-dependent circuit activation and population wide constitutive expression. This document describes the potential of this tool by coupling it to lysis gene, creating the inducible synchronized lysis circuit (iSLC). This circuit enables spatial and temporal modulation of inducer-dependent cargo release for single or multi-strain communities. In particular, this document demonstrates the ability to temporally and spatially control transitions between inactivated population growth, quorum enabled cyclic cargo release and global population death (kill switch). This precise control of circuit activation, the ability for timed strain elimination and the non-toxic nature of p-coumaric acid make this system particularly attractive for potential therapeutic applications in vivo. This document also describes the orthogonality of the iQS system to demonstrate precise control of multi-strain dynamics which has potential to become a key tool for engineering synthetic bacterial communities. The modularity and simplicity of the iQS genetic parts make it straightforward for coupling to any gene of choice, enabling precise spatial and temporal control over population level gene expression.

In one aspect, this disclosure features methods of inducing quorum sensing, the method including: culturing a bacterial strain, where the bacterial strain includes: a first nucleic acid sequence encoding a first activator polypeptide, where expression of the first activator polypeptide results in production of a quorum sensing molecule precursor, where the first nucleic acid sequence is operably linked to a promoter; a second nucleic acid sequence encoding a second activator polypeptide, where expression of the second activator polypeptide results in production of a quorum sensing molecule, where the second nucleic acid sequence is operably linked to a first activatable promoter; a third nucleic acid sequence encoding a third activator polypeptide, where expression of the third activator polypeptide results in activation of the quorum sensing system, where the third nucleic acid sequence is operably linked to a second activatable promoter; and a fourth nucleic acid sequence encoding a gene of interest, where the fourth nucleic acid sequence is operably linked to a third activatable promoter; and contacting the bacterial strain with an inducer molecule; and converting the inducer molecule into a quorum sensing molecule, thereby allowing induction of quorum sensing.

In some embodiments, the quorum sensing system includes a Lux-like quorum sensing system selected from the group consisting of: a rpa system from Rhodopseudomonas palustris; a lux system from Vibrio fischeri; a las system from Pseduomonas aeruginosa; a rhl system from Pseduomonas aeruginosa; a tra system from Agrobacterium tumefaciens; a rpa system from Rhodopseudomonas palustris; an ahy system from Aeromonas hydrophilia; a sma system from Serratia marcescens; cer system from Rhodobacter sphaeroides; and an exp system from Sinorhizobium meliloti. In some embodiments, the quorum sensing system includes all or part of an rpa quorum sensing system, where the rpa sensing system is from Rhodopseudomonas palustris.

In some embodiments, the second activator polypeptide includes a luxI homolog selected from the group consisting of: RpaI, RhlI, BjaI, AubI, CerI, LasI, BraI, SinI, LuxI, and EsaI and where the third activator polypeptide includes a luxR homolog selected from the group consisting of: RpaR, AubR, BjaR, LasR, RhlR, LuxR and TraR.

In some embodiments, (i) the first activatable promoter operably linked to the second nucleic acid is a promoter including one or more lux box-like sequences; (ii) the second activatable promoter operably linked to the third nuleic acid is a promoter including one or more lux box-like sequences; and/or (iii) the third activatable promoter operably linked to the fourth nucleic acid is a promoter including one or more lux box-like sequences.

In some embodiments, the first activator polypeptide includes a p-coumaric acid-CoA ligase, where the p-coumaric acid-CoA ligase is encoded by the 4CL2nt gene from the plant Nicotiana tabacum.

In some embodiments, the quorum sensing molecule precursor is p-Coumaroyl-CoA.

In some embodiments, the second activator polypeptide is a 4-coumaroyl-homoserine lactone synthase (RpaI) synthase, where the RpaI synthase is encoded by the RpaI gene from the plant Rhodopseudomonas palustris.

In some embodiments, the quorum sensing molecule is p-Coumaroyl-HSL.

In some embodiments, the third activator polypeptide includes a signaling molecule, where the signaling molecule interacts with the quorum sensing molecule.

In some embodiments, the signaling molecule is a HTH-type quorum sensing-dependent transcriptional regulator (RpaR), where the RpaR signaling molecule is encoded by the RpaR gene from the plant Rhodopseudomonas palustris.

In some embodiments, the interaction of RpaR and the quorum sensing molecule p-Coumaroyl-HSL results in transcriptional regulation of the fourth nucleic acid sequence.

In some embodiments, the inducer molecule is p-coumaric acid.

In some embodiments, the gene of interest encodes a bacteriophage lytic protein capable of forming a lesion in a host cell's membrane.

In some embodiments, the method further includes one or more additional bacterial strains.

In another aspect, this disclosure features bacterial strains produced by any of the methods described herein.

In another aspect, this disclosure features sets of nucleic acids including any of the nucleic acid sequences described herein.

In another aspect, this disclosure features compositions including any of the bacterial strains that include any of the nucleic acids described herein, any two or more of the bacterial strains described herein, or any of the sets of nucleic acid sequences that include any of the nucleic acid sequences described herein.

In another aspect, this disclosure features a method of treating a disease in a subject, the method including: administering to a subject in need therapeutically effective amounts of any of the bacterial strains described herein or any of the compositions described herein, to thereby treat the disease in the subject. In some embodiments, the disease is cancer.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the disclosure will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows the design and characterization of the p-coumaric acid mediated iQS strain. The chemical structures of the molecules involved in the synthesis of the QS molecule are shown at the top.

FIG. 1B shows a schematic diagram to illustrate predicted dynamics associated to the inducible quorum sensing system as a function of population density and external inducer concentration.

FIG. 1C shows fluorescence microscopy images showing a composite of phase-contrast and GFP fluorescence in microfluidic traps. The data from the raw fluorescence values reflect the iQS dynamics predicted in FIG. 1B.

FIG. 1D shows data from microplate reader experiment obtained by culturing the iQS strain in different p-coumaric acid concentrations. All data points represent mean standard deviation of three independent replicates.

FIG. 1E shows microplate reader experiment data obtained by culturing the wild type E. coli strain in a range p-coumaric acid concentrations. All data points represent mean (solid line) standard deviation of three independent replicates (shaded areas).

FIG. 2A shows schematic diagram illustrating the components of the iSLC strain.

FIG. 2B shows simulations of the mathematical model showing three different dynamics at low (shadding associated with numeral1), medium (shadding associated with numeral 2) and high (shadding associated with numeral 3) inducer values. Medium values are predicted to result in sustained oscillations. Left panel: steady state maximum and minimum cell population values are plotted for a range of inducer concentrations. Right panel: simulated time traces for three representative p-coumaric acid values predict three emergent population dynamics.

FIG. 2C shows comparison between the iSLC and SLC dynamics based upon the model simulations.

FIG. 2D shows a diagram of the microfluidic device used to generate the inducer gradient.

FIG. 2E shows heatmaps representing the fluorescence time traces of all fourteen traps present per column of the device. GFP signal is used as a proxy for population density. Four different inducer conditions are shown: low (1), medium (2), high (3), extra high (4), respectively.

FIG. 2F top panels show representative time series images from the fluorescence channel with three different inducer concentrations: low (0 nM), medium (15 nM), high (110 nM), respectively. FIG. 2F bottom panels show fluorescence time traces plotted together with computer simulations of the mathematical model. For the simulation (dashed) time units are arbitrary, therefore the correspondence is strictly qualitative.

FIG. 3A shows a schematic diagram illustrating the iSLC strain kill switch mechanism.

FIG. 3B shows example time traces (n=104) extracted from the transmitted light channel (grey). Solid black line represent the mean. At time zero the cells were induced with 500 nM p-coumaric acid.

FIG. 3C shows video stills (4×) of the microfluidic chip before (top) and 6 hours after (bottom) induction with 500 nM p-coumaric acid. Left side shows magnified images (30×) of a single representative trap.

FIG. 3D shows cell viability measured by CFU count following addition of the killing signal (p-coumaric acid) in liquid culture. All data points represent mean±standard deviation of three independent replicates.

FIG. 4A shows a schematic diagram illustrating co-culture of the two strains used with orthogonal inducible (iSLC) and non-inducible (SLC) quorum sensing respectively.

FIG. 4B shows heat maps representing the fluorescence time traces of all fourteen traps present per column of the device. Top rows show the GFP values and bottom rows the CFP values. Fluorescence signals are used as a proxy for population density.

FIG. 4C top panels show video stills of the co-culture for three inducer concentrations at multiple time points. FIG. 4C bottom panels show the corresponding fluorescence time traces for GFP and CFP.

FIG. 5 shows iSLC growth curves from plate reader experiments obtained with varying concentrations of p-coumaric acid. Lines and shaded areas represent the mean and standard deviation (n=3) respectively.

FIG. 6A shows a schematic diagram of plasmid maps from strain LEAKY which lacks the Rpa cassette (RpaI-Plux-RpaR).

FIG. 6B shows plate reader data obtained by growing strain LEAKY in presence and absence of glucose respectively. High glucose levels in the cell correspond to low cAMP levels which is involved in the luxI promoter activation due to the presence of a binding site for the CAP-cAMP activating complex.

FIG. 7. shows a schematic diagram of a microfluidic gradient device used for experiments in FIG. 2 and FIG. 4. Inlet 1 is set to zero inducer concentration, while Inlet 2 is set to concentration X to generalize dilution factors.

FIG. 8 shows heat maps of fluorescence data for experiments described in FIG. 2. The approximate p-coumaric acid concentration present in each column is reported at the top. For each column, all fourteen traps are reported.

FIG. 9 shows growth curves from plate reader experiments for the iSLC strain. For all p-coumaric acid concentrations tested the growth curves are unaffected, confirming complete orthogonality to the compound. Lines and shaded areas represent the mean and s.d. (n=3) respectively.

FIG. 10 shows heat maps of fluorescence data for the experiments described in FIG. 4. The approximate p-coumaric acid concentration present in each column is reported at the top. For each column, all fourteen traps are reported. GFP is reported in the top row. CFP is reported in the bottom row.

FIG. 11 shows plasmid maps for plasmids used in this study. LAA stands for LAA ClpXP mediated degradation tag.

FIG. 12 shows simulation of the evolution of all six variables over time. Six pcoumaric acid concentrations are considered to span the emerging population dynamics. Initially, all the variables are set to zero except cell number (N) which is set to 1.

FIG. 13 shows phase portraits of variables N (cell number) and L (lysis protein) showing a Hopf bifurcation caused by the appearance and disappearance of a limit cycle.

DETAILED DESCRIPTION

This document provides compositions and method for inducing quorum sensing in a bacterial stain including culturing a bacterial strain, wherein the bacterial strain comprises: a first nucleic acid sequence encoding a first activator polypeptide, wherein expression of the first activator polypeptide results in production of a quorum sensing molecule precursor, wherein the first nucleic acid sequence is operably linked to a promoter; a second nucleic acid sequence encoding a second activator polypeptide, wherein expression of the second activator polypeptide results in production of a quorum sensing molecule, wherein the second nucleic acid sequence is operably linked to a first activatable promoter; a third nucleic acid sequence encoding a third activator polypeptide that is capable of activating the quorum sensing system, wherein the third nucleic acid sequence is operably linked to a second activatable promoter; and a fourth nucleic acid sequence encoding a gene of interest, wherein the fourth nucleic acid sequence is operably linked to a third activatable promoter; and contacting the bacterial strain with an inducer molecule; and converting the inducer molecule into a quorum sensing molecule, thereby allowing induction of quorum sensing. As used herein, “quorum sensing” can refer to the ability to detect and respond to cell population density by gene regulation.

Also provided herein are any of the isolated nucleic acids encoding any of the activator polypeptides, reporter polypeptides and genes of interest. Also provided herein are the bacterial strains including any of the nucleic acid sequences described herein.

Also provided herein are methods for treating a subject having a disease by administering to the subject a therapeutically effective amount of any of the bacterial strains described herein or any of the pharmaceutical compositions described herein. Additional non-limiting aspects of the bacterial strains, pharmaceutical compositions, kits, and methods are described herein and can be used in any combination without limitation.

This document provides the combination of two pillars of population control: inducibility and cell-to-cell communication. To design this inducible quorum sensing system (iQS), native components of the photosynthetic bacterium Rhodopseudomonas palustris were used, which relies on a plant derived organic compound for the production of its signaling molecule. This inducer, p-coumaric acid, is a ubiquitous molecule present in most fruits and vegetables and has proven to be safe for both bacteria and human cells at relevant concentrations.

The iQS can be coupled with any gene of interest to enable tunable population density-dependent gene expression. This principle was demonstrated by coupling the iQS system to the production of a fluorescent reporter protein in order to characterize the inducible circuit dynamics. Next, as a proof of concept, the iQS was coupled to a lysis gene, creating a tunable platform for cargo release. In direct comparison to non-inducible quorum sensing system, the iQS significantly expands the range of population dynamics, allowing for temporal and spatial control of cargo release and population death. Finally, the orthogonality properties of the iQS system demonstrate the ability to scale up inducibility from the population to the community level. In fact, it has been shown that the quorum sensing molecule produced by the bacterium R. palustris is orthogonal to the majority of well-characterized quorum sensing systems (Lux, Las, Tra, Rhl, Cin), providing a communication channel that can propagate information with minimal signal interference. By co-culturing a two strain community, the ability to control population composition and dynamics was demonstrated by varying inducer concentrations. Overall, the iQS system combines many desirable characteristics into a single genetic circuit: inducibility, tunability, population-level coordination, inducer safety, and orthogonality.

The broad potential of the iQS platform enables a wide range of new functionality for synthetic circuits that rely on cell-to-cell communication systems for population-level coordination. In particular, inducibility broadly expanded the functionality of quorum sensing based circuits by enabling switching between states of circuit quiescence, quorum-dependent circuit activation and population wide constitutive expression. In some embodiments, inducible synchronized lysis circuit (iSLC) enables spatial and temporal modulation of inducer-dependent cargo release for single or multi-strain communities. In particular, iSLC demonstrates the ability to temporally and spatially control transitions between inactivated population growth, quorum enabled cyclic cargo release and global population death (kill switch). This precise control of circuit activation, the ability for timed strain elimination and the non-toxic nature of p-coumaric acid make this system particularly at-tractive for potential therapeutic applications in vivo. Additionally, the iQS system demonstrates precise control of multi-strain dynamics which has potential to become a key tool for engineering synthetic bacterial communities. The main challenge will be the functional expression of the p-coumaric acid-CoA ligase enzyme (4CL2nt) due to its heterologous plant origins.

The modularity and simplicity of the iQS described herein make it straightforward for coupling to any gene of choice, enabling precise spatial and temporal control over population level gene expression. Overall, this work includes multiple technologies from circuit design into microbiological organization, from molecular regulatory mechanisms (synthetic promoters), to single cell protein expression (enzyme catalysis), multi-cellular population coordination (cell-to-cell communication) and multi-species interaction (orthogonal quorum sensing).

The term “a” and “an” refers to one or more (i.e., at least one) of the grammatical object of the article. By way of example, “a cell” encompasses one or more cells.

As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ±20%, ±10%, or ±5%, are within the intended meaning of the recited value.

Unless otherwise specified, a “nucleotide sequence encoding a protein” can include all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.

The term “exogenous” can refer to any material introduced from or originating from outside a cell, a tissue or an organism that is not produced by or does not originate from the same cell, tissue, or organism in which it is being introduced.

The term “transduced”, “transfected”, or “transformed” can refer to a process by which exogenous nucleic acid is introduced or transferred into a cell (e.g., a bacterial cell).

The term “subject” can include any mammal. In some embodiments, the subject is cat, a dog, a goat, a human, a non-human primate, a rodent (e.g., a mouse or a rat), a pig, or a sheep. In some embodiments, the subject has or is at risk of developing a CNS disorder or disease. In some embodiments, the subject has previously been identified or diagnosed as having a CNS disorder or disease.

The term “nucleic acid” can refer to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is DNA. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is RNA.

Modifications can be introduced into a nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., arginine, lysine and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, and tryptophan), nonpolar side chains (e.g., alanine, isoleucine, leucine, methionine, phenylalanine, proline, and valine), beta-branched side chains (e.g., isoleucine, threonine, and valine), and aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine), and aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine).

The term “treating” can refer to a reduction in the number, frequency, severity, or duration of one or more (e.g., two, three, four, five, or six) symptoms of a disease or disorder in a subject (e.g., any of the subjects described herein), and/or results in a decrease in the development and/or worsening of one or more symptoms of a disease or disorder in a subject.

The term “administer” can refer to a method of delivering agents, compounds, or compositions to the desired site of biological action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the compositions described herein are administered intravenously.

The term “promoter” can refer to a DNA sequence recognized by enzymes/proteins in a cell required to initiate the transcription of an operably linked coding sequence (e.g., a nucleic acid encoding an activator polypeptide. A promoter typically refers, to e.g. a nucleotide sequence to which an RNA polymerase and/or any associated factor binds and at which transcription is initiated. The promoter can be constitutive, inducible, or tissue-specific.

The terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, can refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

For sequence comparison of polypeptides, typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment or using publicly available software using known algorithms to achieve maximal alignment. Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.

Quorum Sensing System

In one aspect, this disclosure features an inducible quorum sensing system (iQS) that enables population control of one or more bacterial strains via inducibility and cell-to-cell communication. In some embodiments, the methods include an inducible quorum sensing system that includes: culturing a bacterial strain, wherein the bacterial strain includes: a first nucleic acid sequence encoding a first activator polypeptide that is capable of activating a quorum sensing system, wherein the first nucleic acid sequence is operably linked to a promoter; a second nucleic acid sequence encoding a second activator polypeptide that is capable of activating the quorum sensing system, wherein the second nucleic acid sequence is operably linked to an activatable promoter; a third nucleic acid sequence encoding a third activator polypeptide, wherein expression of the third activator polypeptide results in activation of the quorum sensing system, wherein the third nucleic acid sequence is operably linked to the activatable promoter; and a fourth nucleic acid sequence encoding a gene of interest, wherein the fourth nucleic acid sequence is operably linked to a second activatable promoter; contacting the bacterial strain with an inducer molecule; and converting the inducer molecule into a quorum sensing molecule, thereby allowing induction of quorum sensing.

In some embodiments, the methods include an inducible quorum sensing system that includes: culturing a bacterial strain, wherein the bacterial strain includes: a first nucleic acid sequence encoding a first activator polypeptide that is capable of activating a quorum sensing system, wherein the first nucleic acid sequence is operably linked to a promoter; a second nucleic acid sequence encoding a second activator polypeptide that is capable of activating the quorum sensing system, wherein the second nucleic acid sequence is operably linked to an activatable promoter; and a third nucleic acid sequence encoding a third activator polypeptide that is capable of activating the quorum sensing system, wherein the third nucleic acid sequence is operably linked to an activatable promoter; contacting the bacterial strain with an inducer molecule; and converting the inducer molecule into a quorum sensing molecule, thereby allowing induction of quorum sensing.

In some embodiments, the quorum sensing system is a Lux-like quorum sensing system selected from the group consisting of: a lux system from Vibrio fischeri; a las system from Pseduomonas aeruginosa; a rhl system from Pseduomonas aeruginosa; a tra system from Agrobacterium tumefaciens; a rpa system from Rhodopseudomonas palustris; an ahy system from Aeromonas hydrophilia; a sma system from Serratia marcescens; cer system from Rhodobacter sphaeroides; and an exp system from Sinorhizobium meliloti. In such cases, the method includes a second activator polypeptide and a third activator polypeptide from a Lux-like quorum sensing system.

In some embodiments, the quorum sensing system is based, at least in part, on an acyl-HSL-type quorum sensing system. In some embodiments, an acyl-HSL-type quorum sensing includes luxI- and luxR-type genes.

In some embodiments, a first activator polypeptide includes a p-coumaric acid-CoA ligase, where the p-coumaric acid-CoA ligase is encoded by the 4CL2nt gene from the plant Nicotiana tabacum. In such cases, an inducer molecule can be a p-coumaric acid molecule.

In some embodiments, the inducible quorum sensing system includes a LuxI homolog. In some embodiments, a second activator polypeptide includes a LuxI homolog. LuxI proteins are QS signal synthases that can catalyze amide bond formation between an acyl group on an appropriate side chain donor (most often acyl-acyl carrier protein) and S-adenosylmethionine (SAM) resulting in the final acyl-HSL product. Non-limiting examples of LuxI homologs include: RpaI, RhlI, BjaI, AubI, CerI, LasI, BraI, SinI, LuxI, and EsaL In some embodiments, the inducible quorum sensing system includes a RpaI synthase polypeptide. In some embodiments, the second activator polypeptide is a 4-coumaroyl-homoserine lactone synthase (RpaI) synthase, wherein the RpaI synthase is encoded by the RpaI gene from the plant Rhodopseudomonas palustris.

In some embodiments, the inducible quorum sensing system includes LuxR homologs. In some embodiments, a third activator polypeptide includes a LuxR homolog. LuxR homologs are homodimeric transcription factors, with each monomer consisting of two domains: an N-terminal acyl-HSL binding domain and a C-terminal DNA-binding domain that contains a helix-turn-helix motif. Non-limiting examples of LuxR homologs include: RpaR, AubR, BjaR, LasR, RhlR, LuxR and TraR. In some embodiments, the inducible quorum sensing system includes a RpaR polypeptide. In some embodiments, a third activator polypeptide comprises a signaling molecule, where the signaling molecule is a LuxR homolog (e.g., a RpaR polypeptide).

In some embodiments, a third activator polypeptide is a signaling molecule, where the signaling molecule interacts with the quorum sensing molecule. In some embodiments, a signaling molecule is a HTH-type quorum sensing-dependent transcriptional regulator (RpaR), wherein the RpaR signaling molecule is encoded by the RpaR gene from the plant Rhodopseudomonas palustris.

In some embodiments, the inducible quorum sensing system includes a promoter operably linked to a gene of interest where the promoter can be controlled by a LuxR homolog. In some embodiments, a second activator polypeptide and/or a third activator polypeptide are operably linked to promoters that can be controlled by a LuxR homolog. In some cases, promoters controlled by LuxR homologs can include specific inverted repeat DNA sequences. The inverted DNA repeat elements are known as lux box-like sequences. For example, the lux box is a 20-bp palindromic sequence centered at base pair (bp)—42.5 from the transcription start of the Vibrio fischeri lux operon, which encodes the luminescence functions. With its cognate acyl-HSL, LuxR binds to the lux box and facilitates RNA polymerase binding. In some embodiments, a lux box-lie sequence can be present in promoter operably linked to a gene of interest. In some embodiments, a lux-like box sequence can be present in a promoter operably linked to a second activator polypeptide and/or a third activator polypeptide.

In some embodiments, the quorum sensing system is an rpa (RpaR) quorum sensing system. In some embodiments, the quorum sensing systems is based, at least in part, on a quorum sensing system selected from: Rhodopseudomonas palustris, Bradyrhizobium sp., and Silicibacter pomeroyi. For example, a first activator polypeptide, a second activator polypeptide, and a third activator polypeptide are selected from a quorum sensing system selected from: Rhodopseudomonas palustris, Bradyrhizobium sp., and Silicibacter pomeroyi. In another example, a second activator polypeptide and a third activator polypeptide are selected from a quorum sensing system selected from: Rhodopseudomonas palustris, Bradyrhizobium sp., and Silicibacter pomeroyi.

In some embodiments, a first activator polypeptide is a p-coumaric acid-CoA ligase, the second activator polypeptide is a RpaI synthase, and the third activator polypeptide is a RpaR signaling molecule. In some embodiments, induction of the inducible quorum sensing system is considered to have occurred when the quorum sensing molecule (e.g., the p-coumaroyl-HSL (pC-HSL) molecule) binds to the RpaR signaling molecule. In some cases, the activatable promoter operably linked to a fourth nucleic acid sequence is an activatable promoter that can be activated by a third activator polypeptide (e.g., the RpaR polypeptide) when it is bound to a quorum sensing molecule (e.g., p-coumaroyl-HSL).

In some embodiments, an inducer molecule (e.g., a p-coumaric acid) molecule is used to induce a quorum sensing system. In a non-limiting example, a non-zero p-coumaric acid concentration is converted into an intermediate molecule (e.g., a p-coumaroyl-CoA (pA)) using the enzyme 4CL2nt. The intermediate pA molecule can be referred to as a “quorum sensing molecule precursor.” The intermediate product (pA) is transformed into a quorum sensing molecule (e.g., a p-coumaroyl-HSL (pC-HSL)) using a RpaI synthase enzyme. Once a threshold value of extracellular pC-HSL is reached, intracellular production of luxI driven genes (Real and RpaR) are brought to the ON state, due to the positive feedback provided by the QS promoter (e.g., pLux promoter). In some embodiments, as the p-coumaric acid concentration is increased, the iQS shows a first transition from a stable spiral to a limit cycle which indicates sustained oscillations. A further increase in the inducer molecule concentration causes the limit cycle to disappear in favor of a stable fixed point.

In some embodiments, converting p-coumaric acid to p-coumaroyl-CoA is performed by a first activator polypeptide (e.g., the p-coumaric acid-CoA ligase), where conversion of p-coumaric acid to p-coumaroyl-CoA results in activation of the inducible quorum sensing system. In some embodiments, converting p-coumaroyl-CoA to p-coumaroyl-HSL is performed by a second activator polypeptide (e.g., RpaI synthase), where converstion of p-coumaroyl-CoA to p-courmaroyl-HSL results in activation of the inducible quorum sensing system.

In some embodiments, activation of the inducible quorum sensing system includes binding of a third activator polypeptide (e.g., a RpaR) to a quorum sensing molecule (e.g., a p-coumaroyl-HSL), where binding results in activation of the inducible quorum sensing system. In such cases, binding of a RpaR polypeptide to a p-coumaryol-HSL results in in transcriptional regulation of RpaR-regulatable promoter (e.g., a pLux promoter), where transcriptional regulation of a RpaR-regulatable promoter results in activation of the inducible quorum sensing system. In some cases, binding of a RpaR polypeptide to a p-coumaryol-HSL results in in transcriptional regulation of RpaR-regulatable promoter (e.g., a pLux promoter), where transcriptional regulation a RpaR-regulatable promoter results in activation of the gene of interest.

In some embodiments, the inducible quorum sensing system does not crosstalk with other quorum sensing systems. In such cases, there is no crosstalk between quorum sensing systems when an inducer molecule (e.g., p-coumaric acid) is used to activate the quorum sensing system. This enables the inducible quorum sensing system as described herein to be used with one or more additional quorum sensing systems. For example, the absence of cross-talk between a p-coumaric acid derived signaling molecule and the majority of well-characterized quorum sensing systems, enables the simultaneous use of multiple quorum sensing in co-culture. Non-limiting examples of quorum sensing systems that can be used in conjunction with the inducible quorum sensing system include: Vibrio fischeri; a las system from Pseduomonas aeruginosa; a rhl system from Pseduomonas aeruginosa; a tra system from Agrobacterium tumefaciens; an ahy system from Aeromonas hydrophilia; a sma system from Serratia marcescens; cer system from Rhodobacter sphaeroides; and an exp system from Sinorhizobium meliloti.

In another aspect, this disclosure features a model that accurately predicts three main dynamics of the cell population as the concentration of an inducer molecule (e.g., the p-coumaric acid) is varied. In a non-limiting example, at an inducer concentration of zero, the population grows reaching a steady state value. At small inducer molecule (e.g., p-coumaric acid) concentrations (e.g., <10 nM), the population undergoes small amplitude lysis events followed by steady state. As the p-coumaric acid concentration is increased, the simulations show a first transition from a stable spiral to a limit cycle which indicates sustained oscillations. A further increase in the inducer parameter causes the limit cycle to disappear in favor of a stable fixed point.

In some embodiments, modulating the inducer molecule concentration during the culture enables switching between states of constant growth (circuit quiescence), synchronized oscillations in population density (cyclic cargo release) and inducible population death (kill switch).

In some embodiments, the circuit functionalities of the iQS were demonstrated in the E. Coli strain MG1655, the circuit could potentially be extended to other bacterial species.

Isolated Nucleic Acids

Provided herein are nucleic acids sequences that encode any of the activator polypeptides described herein. Also provided herein are nucleic acids encoding any of the reporter polypeptides described herein. Also provided herein are nucleic acids encoding any of the genes of interests described herein. Also provided herein are expression vectors including any of the nucleic acid sequences described herein. In some embodiments, the expression vectors are multi-cistronic vectors and/or include bidirectional promoters.

In some embodiments of any of the nucleic acid sequences described herein, nucleic acid sequences can include any of the components described in SEQ ID NO: 2-9. In some embodiments, a nucleic acid sequences can include a sequence of any one of SEQ ID NOs: 10-12.

In some embodiments, a second nucleic acid sequence encoding a second activator polypeptide and a third nucleic acid sequence encoding a third activator polypeptide are operably linked to the same promoter (e.g., any of the activatable promoters described herein). For example, the second and third nucleic acid sequences are operably linked to a pLux promoter. In some cases, the second and third nucleic acid sequences are each operably linked to a bi-directional promoter. In some cases, the second and third nucleic acid sequences are operably linked to the same promoter via an internal ribosome entry sequence (IRES) or a self-cleaving 2A peptide. In some embodiments, the second and third nucleic acid sequences are operably linked to different promoters. For example, the second nucleic acid sequence can be linked to a first activatable promoter sequence (e.g., a first pLux promoter), and the third nucleic acid sequence can be linked to a second activatable promoter (e.g., a second pLux promoter).

Also provided herein are vectors including any of the nucleic acids described herein. In some embodiments, the vectors include any of the nucleic acid sequences as described in FIGS. 1A, 2A, 4A, 6A, and 11. The vectors described herein can include any of the combinations of nucleic acid sequences and any of the orientations as described in FIGS. 1A, 2A, 4A, 6A, and 11.

Quorum Sensing Molecule

In some embodiments, a “quorum sensing molecule” can be referred to as a quorum sensing molecule precursor or a quorum sensing molecule. In some cases, a quorum sensing molecule can be any appropriate quorum sensing molecule. In some embodiments, the quorum-sensing molecule can be an N-acyl homoserine lactone (AHL). In some embodiments, the quorum-sensing molecule can be a homoserine lactone (HSL) or a homolog thereof. For example, the quorum-sensing molecule can be p-Coumaroyl-HSL. In some embodiments, a quorum-sensing molecule can be an auto-inducing peptide (AIP) from a Gram-positive bacteria. Other quorum-sensing molecules are known in the art.

4CL2nt Gene

In some embodiments, the method includes a nucleic acid sequence encoding a p-coumaric acid-CoA ligase polypeptide. In some cases, a first activator polypeptide is a p-coumaric acid-CoA ligase. The p-coumaric acid-CoA ligase converts p-coumaric acid to p-coumaroyl-CoA. In some embodiments, the nucleic acid sequence encoding a p-coumaric acid-CoA ligase polypeptide is a 4CL2nt gene from the plant Nicotiana tabacum.

In some embodiments, the 4CL2nt polypeptide is encoded by a nucleic acid sequence that is at least 70% identical (e.g., at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 1. In some embodiments, the nucleic acid sequence that encodes the 4CL2nt polypeptide can be a codon-optimized version of SEQ ID NO: 1.

In some embodiments, the nucleic acid sequence that encodes the 4CL2nt polypeptide is operably linked to a promoter. In some embodiments, the promoter is an activatable promoter (e.g., any of the exemplary activatable promoters described herein). In some embodiments, the nucleic acid sequence encoding the 4CL2nt polypeptide is operably linked to a promoter having a nucleic acid sequence that is at least 70% identical (e.g., at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 2. In some embodiments, the promoter operably linked to the nucleic acid sequence encoding the 4CL2nt polypeptide is referred to as J23106.

Activatable Promoter

An activatable promoter can be any appropriate activatable promoter. In some embodiments, the activatable promoter can be activated, directly or indirectly, by the quorum-sensing molecule. In this way, in some embodiments, a feedback loop can be set up such that the presence of the quorum-sensing molecule drives accumulation of the quorum-sensing molecule. For example, binding of the quorum sensing molecule to a third activator polypeptide (e.g., the RpaR polypeptide) can result in activation of an activatable promoter (e.g., any of the activatable promoters described herein) operably linked to any of the activator polypeptides or genes of interest described herein. In some cases, the feedback loop is a positive feedback loop for a third activator polypeptide. For example, binding of the quorum sensing molecule to the third activator polypeptide results in activation of the promoter operably linked to the third activator polypeptide. In some cases, the feedback loop is a positive feedback loop for a second activator polypeptide (e.g., the RpaI synthase polypeptide) that converts p-coumaroyl-CoA to p-coumaroyl-HSL. In such cases, binding of the quorum sensing molecule to the third activator polypeptide results in activation of the promoter operably linked to a second activator polypeptide (e.g., the RpaI synthase polypeptide). In some cases, the feedback loop is a positive feedback loop for a gene of interest encoded by a fourth nucleic acid. In such cases, binding of the quorum sensing molecule to the third activator polypeptide results in activation of a promoter operably linked to the gene of interest.

In some embodiments, the first activatable promoter includes the same sequence as the second activatable promoter. For example, the first activatable promoter and the second activatable promoter include a sequence including one or more lux box-like sequences. In some embodiments, the first activatable promoter and the second activatable promoter are the same promoter sequence (e.g., an activatable promoter sequence that enables bidirectional expression). In some embodiments, the first activatable promoter, the second activatable promoter, and the third activatable promoter each include the same sequence. In a non-limiting example, the first activatable promoter, the second activatable promoter, and the third activatable promoter each include a sequence including one or more lux box-like sequences.

In some embodiments of any of the bacterial strains described herein, the activatable promoter is an AHL-activatable promoter. In some embodiments of any of the bacterial strains described herein, the activatable promoter is a LuxR-AHL activatable luxI promoter. In some embodiments, the activatable promoter is a RpaR-AHL activatable RpaI promoter. In some embodiments, the activatable promoter is a TraR-HSL activatable traI promoter. Other activatable promoters are known in the art.

In some embodiments, the quorum-sensing molecule is an AHL, the activatable promoter is a LuxR-AHL activatable luxI promoter, and the activator polypeptide is a LuxI. In some embodiments, the quorum-sensing molecule is an AHL, the activatable promoter is a RpaR-AHL activatable RpaI promoter, and the activator polypeptide is a RpaI. In some embodiments, the quorum-sensing molecule is a HSL, the activatable promoter is a TraR-HSL activatable traI promoter, and the activator polypeptide is a Trai. In some embodiments, the quorum-sensing molecule is a HSL, the activatable promoter is a LuxR-AHL activatable luxI promoter, and the activator polypeptide is a RpaI.

In some embodiments, the promoter can be an activatable promoter. In some embodiments, the promoter can be a promoter that is activated, directly or indirectly, by the quorum sensing molecule. In some embodiments, the promoter can be an activatable promoter as described above. In some embodiments, the promoter can be a constitutive promoter. Many constitutive promoters are known in the art.

p-Coumaric Acid

In some embodiments of any of the methods described herein, the inducer molecule includes a p-coumaric acid molecule. In some embodiments, the concentration of an inducer molecule contacted to the one or more bacterial strains determines the level of induction of the inducible quorum sensing system in the one or more bacterial strains. In some embodiments, the concentration of an inducer molecule contacted to the one or more bacterial strains results in no induction of the inducible quorum sensing system. In some cases, where there is no induction of the inducible quorum sensing system, the quorum sensing system is considered to be “OFF.” In some embodiments, the concentration of an inducer molecule contacted to the one or more bacterial strains results in “intermediate” induction of the inducible quorum sensing system. As used herein, “intermediate” can refer to induction of the inducible quorum sensing system that occurs in the presence of a non-zero concentration of inducer molecule. In some embodiments, the concentration of an inducer molecule contacted to the one or more bacterial strains results in induction of the inducible quorum sensing system. In some cases, where there is induction of the inducible quorum sensing system, the inducible quorum sensing system is considered to be “ON.”

In some embodiments, the inducer molecule (e.g., p-coumaric acid) that is contacted with the one or more biological strains includes a concentration of between about 1 nM to about 1000 nM (e.g., about 1 nM to about 900 nM, about 1 nM to about 800 nM, about 1 nM to about 700 nM, about 1 nM to about 600 nM, about 1 nM to about 500 nM, about 1 nM to about 400 nM, about 1 nM to about 300 nM, about 1 nM to about 200 nM, about 1 nM to about 100 nM, about 1 nM to about 90 nM, about 1 nM to about 80 nM, about 1 nM to about 70 nM, about 1 nM to about 60 nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about 1 nM to about 30 nM, about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 1000 nM, about 2 nM to about 900 nM, about 2 nM to about 800 nM, about 2 nM to about 700 nM, about 2 nM to about 600 nM, about 2 nM to about 500 nM, about 2 nM to about 400 nM, about 2 nM to about 300 nM, about 2 nM to about 200 nM, about 2 nM to about 100 nM, about 2 nM to about 90 nM, about 2 nM to about 80 nM, about 2 nM to about 70 nM, about 2 nM to about 60 nM, about 2 nM to about 50 nM, about 2 nM to about 40 nM, about 2 nM to about 30 nM, about 2 nM to about 20 nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM, about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about 1000 nM, about 3 nM to about 900 nM, about 3 nM to about 800 nM, about 3 nM to about 700 nM, about 3 nM to about 600 nM, about 3 nM to about 500 nM, about 3 nM to about 400 nM, about 3 nM to about 300 nM, about 3 nM to about 200 nM, about 3 nM to about 100 nM, about 3 nM to about 90 nM, about 3 nM to about 80 nM, about 3 nM to about 70 nM, about 3 nM to about 60 nM, about 3 nM to about 50 nM, about 3 nM to about 40 nM, about 3 nM to about 30 nM, about 3 nM to about 20 nM, about 3 nM to about 10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM to about 7 nM, about 3 nM to about 6 nM, about 3 nM to about 5 nM, about 3 nM to about 4 nM, about 4 nm to about 1000 nM, about 4 nM to about 900 nM, about 4 nM to about 800 nM, about 4 nM to about 700 nM, about 4 nM to about 600 nM, about 4 nM to about 500 nM, about 4 nM to about 400 nM, about 4 nM to about 300 nM, about 4 nM to about 200 nM, about 4 nM to about 100 nM, about 4 nM to about 90 nM, about 4 nM to about 80 nM, about 4 nM to about 70 nM, about 4 nM to about 60 nM, about 4 nM to about 50 nM, about 4 nM to about 40 nM, about 4 nM to about 30 nM, about 4 nM to about 20 nM, about 4 nM to about 10 nM, about 4 nM to about 9 nM, about 4 nM to about 8 nM, about 4 nM to about 7 nM, about 4 nM to about 6 nM, about 4 nM to about 5 nM, about 5 nM to about 1000 nM, about 5 nM to about 900 nM, about 5 nM to about 800 nM, about 5 nM to about 700 nM, about 5 nM to about 600 nM, about 5 nM to about 500 nM, about 5 nM to about 400 nM, about 5 nM to about 300 nM, about 5 nM to about 200 nM, about 5 nM to about 100 nM, about 5 nM to about 90 nM, about 5 nM to about 80 nM, about 5 nM to about 70 nM, about 5 nM to about 60 nM, about 5 nM to about 50 nM, about 5 nM to about 40 nM, about 5 nM to about 30 nM, about 5 nM to about 20 nM, about 5 nM to about 10 nM, about 5 nM to about 9 nM, about 5 nM to about 8 nM, about 5 nM to about 7 nM, about 5 nM to about 6 nM, about 6 nM to about 1000 nM, about 6 nM to about 900 nM, about 6 nM to about 800 nM, about 6 nM to about 700 nM, about 6 nM to about 600 nM, about 6 nM to about 500 nM, about 6 nM to about 400 nM, about 6 nM to about 300 nM, about 6 nM to about 200 nM, about 6 nM to about 100 nM, about 6 nM to about 90 nM, about 6 nM to about 80 nM, about 6 nM to about 70 nM, about 6 nM to about 60 nM, about 6 nM to about 50 nM, about 6 nM to about 40 nM, about 6 nM to about 30 nM, about 6 nM to about 20 nM, about 6 nM to about 10 nM, about 6 nM to about 9 nM, about 6 nM to about 8 nM, about 6 nM to about 7 nM, about 7 nM to about 1000 nM, about 7 nM to about 900 nM, about 7 nM to about 800 nM, about 7 nM to about 700 nM, about 7 nM to about 600 nM, about 7 nM to about 500 nM, about 7 nM to about 400 nM, about 7 nM to about 300 nM, about 7 nM to about 200 nM, about 7 nM to about 100 nM, about 7 nM to about 90 nM, about 7 nM to about 80 nM, about 7 nM to about 70 nM, about 7 nM to about 60 nM, about 7 nM to about 50 nM, about 7 nM to about 40 nM, about 7 nM to about 30 nM, about 7 nM to about 20 nM, about 7 nM to about 10 nM, about 7 nM to about 9 nM, about 7 nM to about 8 nM, about 8 nM to about 1000 nM, about 8 nM to about 900 nM, about 8 nM to about 800 nM, about 8 nM to about 700 nM, about 8 nM to about 600 nM, about 8 nM to about 500 nM, about 8 nM to about 400 nM, about 8 nM to about 300 nM, about 8 nM to about 200 nM, about 8 nM to about 100 nM, about 8 nM to about 90 nM, about 8 nM to about 80 nM, about 8 nM to about 70 nM, about 8 nM to about 60 nM, about 8 nM to about 50 nM, about 8 nM to about 40 nM, about 8 nM to about 30 nM, about 8 nM to about 20 nM, about 8 nM to about 10 nM, about 8 nM to about 9 nM, about 9 nM to about 1000 nM, about 9 nM to about 900 nM, about 9 nM to about 800 nM, about 9 nM to about 700 nM, about 9 nM to about 600 nM, about 9 nM to about 500 nM, about 9 nM to about 400 nM, about 9 nM to about 300 nM, about 9 nM to about 200 nM, about 9 nM to about 100 nM, about 9 nM to about 90 nM, about 9 nM to about 80 nM, about 9 nM to about 70 nM, about 9 nM to about 60 nM, about 9 nM to about 50 nM, about 9 nM to about 40 nM, about 9 nM to about 30 nM, about 9 nM to about 20 nM, about 9 nM to about 10 nM, about 10 nM to about 1000 nM, about 10 nM to about 900 nM, about 10 nM to about 800 nM, about 10 nM to about 700 nM, about 10 nM to about 600 nM, about 10 nM to about 500 nM, about 10 nM to about 400 nM, about 10 nM to about 300 nM, about 10 nM to about 200 nM, about 10 nM to about 100 nM, about 10 nM to about 90 nM, about 10 nM to about 80 nM, about 10 nM to about 70 nM, about 10 nM to about 60 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 10 nM to about 20 nM, about 20 nM to about 1000 nM, about 20 nM to about 900 nM, about 20 nM to about 800 nM, about 20 nM to about 700 nM, about 20 nM to about 600 nM, about 20 nM to about 500 nM, about 20 nM to about 400 nM, about 20 nM to about 300 nM, about 20 nM to about 200 nM, about 20 nM to about 100 nM, about 20 nM to about 90 nM, about 20 nM to about 80 nM, about 20 nM to about 70 nM, about 20 nM to about 60 nM, about 20 nM to about 50 nM, about 20 nM to about 40 nM, about 20 nM to about 30 nM, about 30 nM to about 1000 nM, about 30 nM to about 900 nM, about 30 nM to about 800 nM, about 30 nM to about 700 nM, about 30 nM to about 600 nM, about 30 nM to about 500 nM, about 30 nM to about 400 nM, about 30 nM to about 300 nM, about 30 nM to about 200 nM, about 30 nM to about 100 nM, about 30 nM to about 90 nM, about 30 nM to about 80 nM, about 30 nM to about 70 nM, about 30 nM to about 60 nM, about 30 nM to about 50 nM, about 30 nM to about 40 nM, about 40 nM to about 1000 nM, about 40 nM to about 900 nM, about 40 nM to about 800 nM, about 40 nM to about 700 nM, about 40 nM to about 600 nM, about 40 nM to about 500 nM, about 40 nM to about 400 nM, about 40 nM to about 300 nM, about 40 nM to about 200 nM, about 40 nM to about 100 nM, about 40 nM to about 90 nM, about 40 nM to about 80 nM, about 40 nM to about 70 nM, about 40 nM to about 60 nM, about 40 nM to about 50 nM, about 50 nM to about 1000 nM, about 50 nM to about 900 nM, about 50 nM to about 800 nM, about 50 nM to about 700 nM, about 50 nM to about 600 nM, about 50 nM to about 500 nM, about 50 nM to about 400 nM, about 50 nM to about 300 nM, about 50 nM to about 200 nM, about 50 nM to about 100 nM, about 50 nM to about 90 nM, about 50 nM to about 80 nM, about 50 nM to about 70 nM, about 50 nM to about 60 nM, about 60 nM to about 1000 nM, about 60 nM to about 900 nM, about 60 nM to about 800 nM, about 60 nM to about 700 nM, about 60 nM to about 600 nM, about 60 nM to about 500 nM, about 60 nM to about 400 nM, about 60 nM to about 300 nM, about 60 nM to about 200 nM, about 60 nM to about 100 nM, about 60 nM to about 90 nM, about 60 nM to about 80 nM, about 60 nM to about 70 nM, about 70 nM to about 1000 nM, about 70 nM to about 900 nM, about 70 nM to about 800 nM, about 70 nM to about 700 nM, about 70 nM to about 600 nM, about 70 nM to about 500 nM, about 70 nM to about 400 nM, about 70 nM to about 300 nM, about 70 nM to about 200 nM, about 70 nM to about 100 nM, about 70 nM to about 90 nM, about 70 nM to about 80 nM, about 80 nM to about 1000 nM, about 80 nM to about 900 nM, about 80 nM to about 800 nM, about 80 nM to about 700 nM, about 80 nM to about 600 nM, about 80 nM to about 500 nM, about 80 nM to about 400 nM, about 80 nM to about 300 nM, about 80 nM to about 200 nM, about 80 nM to about 100 nM, about 80 nM to about 90 nM, about 90 nM to about 1000 nM, about 90 nM to about 900 nM, about 90 nM to about 800 nM, about 90 nM to about 700 nM, about 90 nM to about 600 nM, about 90 nM to about 500 nM, about 90 nM to about 400 nM, about 90 nM to about 300 nM, about 90 nM to about 200 nM, about 90 nM to about 100 nM, about 100 nM to about 1000 nM, about 100 nM to about 900 nM, about 100 nM to about 800 nM, about 100 nM to about 700 nM, about 100 nM to about 600 nM, about 100 nM to about 500 nM, about 100 nM to about 400 nM, about 100 nM to about 300 nM, about 100 nM to about 200 nM, about 200 nM to about 1000 nM, about 200 nM to about 900 nM, about 200 nM to about 800 nM, about 200 nM to about 700 nM, about 200 nM to about 600 nM, about 200 nM to about 500 nM, about 200 nM to about 400 nM, about 200 nM to about 300 nM, about 300 nM to about 1000 nM, about 300 nM to about 900 nM, about 300 nM to about 800 nM, about 300 nM to about 700 nM, about 300 nM to about 600 nM, about 300 nM to about 500 nM, about 300 nM to about 400 nM, about 400 nM to about 1000 nM, about 400 nM to about 900 nM, about 400 nM to about 800 nM, about 400 nM to about 700 nM, about 400 nM to about 600 nM, about 400 nM to about 500 nM, about 500 nM to about 1000 nM, about 500 nM to about 900 nM, about 500 nM to about 800 nM, about 500 nM to about 700 nM, about 500 nM to about 600 nM, about 600 nM to about 1000 nM, about 600 nM to about 900 nM, about 600 nM to about 800 nM, about 600 nM to about 700 nM, about 700 nM to about 1000 nM, about 700 nM to about 900 nM, about 700 nM to about 800 nM, about 800 nM to about 1000 nM, about 800 nM to about 900 nM, or about 900 nM to about 1000 nM). In some embodiments, the concentration of inducer molecule (e.g., p-coumaric acid) that results in induction of the inducible quorum sensing system is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM or more.

In some embodiments, the inducer molecule concentration can be modulated during the culture of any one of the one or more bacterial strains. For example, the culture can include a first concentration for a first period of time and a second concentration for a second period of time. In some embodiments, the cultures can include a third concentration for a third period of time. In some embodiments, the cultures can include a fourth concentration for a fourth period of time. Modulating the inducer molecule concentration during the culture enables switching between states of constant growth (circuit quiescence), synchronized oscillations in population density (cyclic cargo release) and inducible population death (kill switch).

In some embodiments, the concentration of the inducer molecule (e.g., p-coumaric acid) that induces the quorum sensing system (e.g., quorum sensing system is considered in the “ON” state) when administered is between about 15 nM to about 1000 nM (or any of the subranges described herein).

In some embodiments, the concentration of the inducer molecule (e.g., p-coumaric acid) that results in intermediate induction of the quorum sensing system is between about 0.1 nM to about 1 nM (e.g., about 0.1 nM to about 0.9 nM, about 0.1 nM to about 0.8 nM, about 0.1 nM to about 0.7 nM, about 0.1 nM to about 0.6 nM, about 0.1 nM to about 0.7 nM, about 0.1 nM to about 0.6 nM, about 0.1 nM to about 0.5 nM, about 0.1 nM to about 0.4 nM, about 0.1 nM to about 0.3 nM, about 0.1 nM to about 0.2 nM, about 0.2 nM to about 1 nM, about 0.2 nM to about 0.9 nM, about 0.2 nM to about 0.8 nM, about 0.2 nM to about 0.7 nM, about 0.2 nM to about 0.6 nM, about 0.2 nM to about 0.7 nM, about 0.2 nM to about 0.6 nM, about 0.2 nM to about 0.5 nM, about 0.2 nM to about 0.4 nM, about 0.2 nM to about 0.3 nM, about 0.3 nM to about 1 nM, about 0.3 nM to about 0.9 nM, about 0.3 nM to about 0.8 nM, about 0.3 nM to about 0.7 nM, about 0.3 nM to about 0.6 nM, about 0.3 nM to about 0.7 nM, about 0.3 nM to about 0.6 nM, about 0.3 nM to about 0.5 nM, about 0.3 nM to about 0.4 nM, about 0.4 nM to about 1 nM, about 0.4 nM to about 0.9 nM, about 0.4 nM to about 0.8 nM, about 0.4 nM to about 0.7 nM, about 0.4 nM to about 0.6 nM, about 0.4 nM to about 0.7 nM, about 0.4 nM to about 0.6 nM, about 0.4 nM to about 0.5 nM, about 0.5 nM to about 1 nM, about 0.5 nM to about 0.9 nM, about 0.5 nM to about 0.8 nM, about 0.5 nM to about 0.7 nM, about 0.5 nM to about 0.6 nM, about 0.6 nM to about 1 nM, about 0.6 nM to about 0.9 nM, about 0.6 nM to about 0.8 nM, about 0.6 nM to about 0.7 nM, about 0.7 nM to about 1 nM, about 0.7 nM to about 0.9 nM, about 0.7 nM to about 0.8 nM, about 0.8 nM to about 1 nM, about 0.8 nM to about 0.9 nM, or about 0.9 nM to about 1 nM).

In some embodiments, the one or more bacterial strains are contacted with an inducer molecule (e.g., a p-coumaric acid) for about 1 hour to about 60 hours (e.g., about 1 hour to about 58 hours, about 1 hour to about 56 hours, about 1 hour to about 54 hours, about 1 hour to about 52 hours, about 1 hour to about 50 hours, about 1 hour to about 48 hours, about 1 hour to about 46 hours, about 1 hour to about 44 hours, about 1 hour to about 42 hours, about 1 hour to about 40 hours, about 1 hour to about 38 hours, about 1 hour to about 36 hours, about 1 hour to about 34 hours, about 1 hour to about 32 hours, about 1 hour to about 30 hours, about 1 hour to about 28 hours, about 1 hour to about 26 hours, about 1 hour to about 24 hours, about 1 hour to about 22 hours, about 1 hour to about 20 hours, about 1 hour to about 18 hours, about 1 hour to about 16 hours, about 1 hour to about 14 hours, about 1 hour to about 12 hours, about 1 hour to about 10 hours, about 1 hour to about 8 hours, about 1 hour to about 6 hours, about 1 hour to about 4 hours, about 1 hour to about 2 hours, about 2 hours to about 32 hours, about 2 hours to about 30 hours, about 2 hours to about 28 hours, about 2 hours to about 26 hours, about 2 hours to about 24 hours, about 2 hours to about 22 hours, about 2 hours to about 20 hours, about 2 hours to about 18 hours, about 2 hours to about 16 hours, about 2 hours to about 14 hours, about 2 hours to about 12 hours, about 2 hours to about 10 hours, about 2 hours to about 8 hours, about 2 hours to about 6 hours, about 2 hours to about 4 hours, about 4 hours to about 32 hours, about 4 hours to about 30 hours, about 4 hours to about 28 hours, about 4 hours to about 26 hours, about 4 hours to about 24 hours, about 4 hours to about 22 hours, about 4 hours to about 20 hours, about 4 hours to about 18 hours, about 4 hours to about 16 hours, about 4 hours to about 14 hours, about 4 hours to about 12 hours, about 4 hours to about 10 hours, about 4 hours to about 8 hours, about 4 hours to about 6 hours, about 6 hours to about 32 hours, about 6 hours to about 30 hours, about 6 hours to about 28 hours, about 6 hours to about 26 hours, about 6 hours to about 24 hours, about 6 hours to about 22 hours, about 6 hours to about 20 hours, about 6 hours to about 18 hours, about 6 hours to about 16 hours, about 6 hours to about 14 hours, about 6 hours to about 12 hours, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 32 hours, about 8 hours to about 30 hours, about 8 hours to about 28 hours, about 8 hours to about 26 hours, about 8 hours to about 24 hours, about 8 hours to about 22 hours, about 8 hours to about 20 hours, about 8 hours to about 18 hours, about 8 hours to about 16 hours, about 8 hours to about 14 hours, about 8 hours to about 12 hours, about 8 hours to about 10 hours, about 10 hours to about 32 hours, about 10 hours to about 30 hours, about 10 hours to about 28 hours, about 10 hours to about 26 hours, about 10 hours to about 24 hours, about 10 hours to about 22 hours, about 10 hours to about 20 hours, about 10 hours to about 18 hours, about 10 hours to about 16 hours, about 10 hours to about 14 hours, about 10 hours to about 12 hours, about 12 hours to about 32 hours, about 12 hours to about 30 hours, about 12 hours to about 28 hours, about 12 hours to about 26 hours, about 12 hours to about 24 hours, about 12 hours to about 22 hours, about 12 hours to about 20 hours, about 12 hours to about 18 hours, about 12 hours to about 16 hours, about 12 hours to about 14 hours, about 14 hours to about 32 hours, about 14 hours to about 30 hours, about 14 hours to about 28 hours, about 14 hours to about 26 hours, about 14 hours to about 24 hours, about 14 hours to about 22 hours, about 14 hours to about 20 hours, about 14 hours to about 18 hours, about 14 hours to about 16 hours, about 16 hours to about 32 hours, about 16 hours to about 30 hours, about 16 hours to about 28 hours, about 16 hours to about 26 hours, about 16 hours to about 24 hours, about 16 hours to about 22 hours, about 16 hours to about 20 hours, about 16 hours to about 18 hours, about 18 hours to about 32 hours, about 18 hours to about 30 hours, about 18 hours to about 28 hours, about 18 hours to about 26 hours, about 18 hours to about 24 hours, about 18 hours to about 22 hours, about 18 hours to about 20 hours, about 20 hours to about 32 hours, about 20 hours to about 30 hours, about 20 hours to about 28 hours, about 20 hours to about 26 hours, about 20 hours to about 24 hours, about 20 hours to about 22 hours, about 22 hours to about 32 hours, about 22 hours to about 30 hours, about 22 hours to about 28 hours, about 22 hours to about 26 hours, about 22 hours to about 24 hours, about 24 hours to about 32 hours, about 24 hours to about 30 hours, about 24 hours to about 28 hours, about 24 hours to about 26 hours, about 26 hours to about 32 hours, about 26 hours to about 30 hours, about 26 hours to about 28 hours, about 28 hours to about 32 hours, about 28 hours to about 30 hours, about 30 hours to about 32 hours, about 24 hour to about 50 hours, about 30 hour to about 50 hours, about 36 hour to about 50 hours, about 24 hour to about 48 hours, about 24 hour to about 36 hours, or about 36 hour to about 48 hours). In some embodiments, the method includes contacting the one or more bacterial strains with an inducer molecule (e.g., p-coumaric acid) for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 or more days.

Synchronized Lysis Circuit

In some embodiments, any of the bacterial strains described herein can produce a therapeutic protein or a suite of therapeutic proteins, the expression of which can be controlled by a genetic circuit such as a Synchronized Lysis Circuit (SLC). In some embodiments, exemplary SLC's can include those described in International Application No. PCT/US18/33555, which is incorporated herein by reference in its entirety. The quorum driven synchronized lysis circuit (SLC) has been investigated for microbial therapeutics such as drug delivery to tumors in vivo. With the SLC circuit, a quorum sensing molecule (AHL), gradually accumulates in the growth environment in proportion to population density. When the population reaches a threshold density, synchronized lysis eliminates around 90% of the bacteria, leaving about 10% to reseed population growth. The resulting dynamics of the cell population are cycles of cell growth and synchronized lysis. However, when employed in in vivo environments where selective media cannot be used, plasmid loss or mutations are expected to result in loss of function over long time periods. In some embodiment, a circuit designed for monocultures, such as the SLC, can have drastically broadened use-cases when expanded into the setting of a community. In some embodiments, the methods include orthogonal co-lysis or “ortholysis” systems, methods, and compositions that can be used for in situ drug delivery systems. In some embodiments, this phenomenon of stably co-culturing two metabolically competitive strains through orthogonal self-lysing offers the possibility of many unique applications beyond drug delivery where the use of synthetic microbial ecosystems is advantageous.

In some embodiments, the gene of interest encodes a bacteriophage lytic protein capable of forming a lesion in a host cell's membrane. A non-limiting example of a gene of interest that is capable of forming a lesion in a host cell's membrane includes the bacteriophage ΦX174 gene. In some embodiments, the binding of the quorum sensing molecule to the third activator polypeptide results in activation of the promoter operably linked to the gene of interest. For example, the binding of p-coumaroyl-HSL to the RpaR polypeptide results in activation of the promoter operably linked to the nucleic acid sequence encoding bacteriophage ΦX174.

Gene of Interest

In some embodiments, the method includes one or more bacterial strains having a fourth nucleic acid encoding a gene of interest. In some embodiments, the gene of interest is a therapeutic gene product. In some embodiments, a therapeutic gene product can be a therapeutic agent. In some embodiments, a therapeutic agent can be any of the therapeutic agents described herein. For example, in some embodiments, a therapeutic agent can be selected from the group consisting of: an inhibitory nucleic acid (e.g., siRNA, shRNA, miRNA, or antisense (e.g., antisense DNA, antisense RNA, or a synthetic analog)), a cytokine, an enzyme, a peptide hormone, a fusion protein, a clotting factor, and an antibody or antigen-binding fragment thereof. In some embodiments, an additional nucleic acid and/or protein (e.g., a heterologous nucleic acid and/or protein) may be secreted, exocytosed, or otherwise exported from a bacterial strain as described herein. In some embodiments, an additional nucleic acid and/or protein (e.g., a heterologous nucleic acid and/or protein) may be released upon lysis of a bacterial strain as described herein. In some embodiments, an additional nucleic acid and/or protein (e.g., a heterologous nucleic acid and/or protein) may be displayed on the surface a bacterial strain as described herein.

In some instances, the therapeutic agent is a therapeutic polypeptide. In some instances, the therapeutic polypeptide includes one or more polypeptides (e.g., 2, 3, 4, 5, or 6). In some instances, the therapeutic polypeptide is conjugated to a toxin, a radioisotope, or a drug via a linker (e.g., a cleavable linker, a non-cleavable linker).

In some instances, the therapeutic agent is cytotoxic or cytostatic to a target cell. The phrase “cytotoxic to a target cell” refers to the inducement, directly or indirectly, in the death (e.g., necrosis or apoptosis) of the target cell. For example, a target cell can be a cancer cell (e.g., a cancerous cell or a tumor-associated immune cell (e.g., macrophage) or an infected cell.

Toxin/Antitoxin System

In some embodiments, a vector includes a vector-stabilizing element. For example, a vector stabilizing element can be part of the toxin/antitoxin system. In some embodiments, the toxin/antitoxin system may be replaced by a bacteriocin/immunity protein system. As used herein, the term “toxin/antitoxin” includes both the toxin/antitoxin systems described herein above, including bacteriocin/immunity protein systems. Bacteriocins are ribosomally-synthesized peptides that are produced by bacteria. Bacteriocins are non-toxic to bacteria that produce the bacteriocins and are generally toxic to other bacteria. Typically, a bacterium that produces a bacteriocin also produces an immunity protein that can inhibit or prevent the toxic effect of the bacteriocin. Accordingly, a bacteriocin and a corresponding immunity protein can be used in an analogous fashion to a toxin/antitoxin system as described herein. Most bacteriocins are extremely potent, and exhibit antimicrobial activity at nanomolar concentrations. By way of example, eukaryotic produced microbials have 10² to 10³ lower activities.

Non-limiting examples of bacteriocins that can be included in any of the bacteria strains, systems and methods described herein include: acidocin, actagardine, agrocin, alveicin, aureocin, aureocin A53, aureocin A70, bisin, carnocin, carnocyclin, caseicin, cerein, circularin A, colicin, curvaticin, divercin, duramycin, enterocin, enterolysin, epidermin/gallidermin, erwiniocin, gardimycin, gassericin A, glycinecin, halocin, klebicin, lactosin S, lactococcin, lacticin, leucoccin, lysostaphin, macedocin, mersacidin, mesentericin, microbisporicin, microcin S, mutacin, nisin, paenibacillin, planosporicin, pediocin, pentocin, plantaricin, pneumocyclicin, pyocin, reutericin 6, sakacin, salivaricin, sublancin, subtilin, sulfolobicin, tasmancin, thuricin 17, trifolitoxin, variacin, vibriocin, warnericin, cytolisin, pyocyn S2, colicin A, colicin El, microcin MccE492, and warnerin.

In some embodiments, the bacteriocin is obtained from a Gram negative bacteria (e.g., microcins (e.g., microcin V of E. coli, subtilosin A from B. subtillis), colicins (E.g., colicin produced by and toxic to certain strains of E. coli (e.g., colicin A, colicin B, colicin El, colicin E3, colicin E5, and colicin E7), tailocins (e.g., R-type pyocins, F-type pyocins)).

In some embodiments, the bacteriocin is obtained from a Gram positive bacteria (e.g., class I bacteriocins (e.g., Nisin, lantibiotics), class II bacteriocins (e.g., IIa pediocin-like bacteriocins, IIb bacteriocins (e.g., lactococcin G), IIc cyclic peptides (e.g., enterocin AS-48), IId single peptide bacteriocins (e.g., aureocin A53), class III bacteriocins (e.g., Ma (e.g., bacteriolysins), and IIIb (which kill the target by disrupting the membrane potential), or class IV bacteriocins (e.g., complex bacteriocins containing lipid or carbohydrate moieties)).

Methods of Introducing a Nucleic Acid in a Cell

Any of the isolated nucleic acids described herein can be introduced into any cell, e.g., a bacterial cell. Methods of introducing nucleic acids and expression vectors into a bacterial cell are known in the art. For example, transformation can be used to introduce a nucleic acid into a bacterial cell.

Culturing Bacterial Strains

Methods of culturing bacterial cells are well known in the art. Cells can be maintained in vitro under conditions that favor cell proliferation, cell growth, and/or cell differentiation. For example, cells can be cultured by contacting a cell (e.g., any of the cells described herein) with a cell culture medium that includes supplemental growth factors to support cell viability and cell growth.

In some embodiments, the culturing or co-culturing occurs in a microfluidic device. In some embodiments, the culturing or co-culturing occurs in a cell culture vessel (e.g., a cell culture plate, a bioreactor) (see PCT/US2018/213815, herein incorporated by reference in its entirety).

Also provided herein are methods of co-culturing at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) bacterial strains (e.g., any of the bacterial strains described herein). In some embodiments, a ratio of inoculation of at least one bacterial strain to at least one other bacterial strain is between 100000:1 and 1: 100000 (e.g., 100000: 1, 95000: 1, 90000: 1, 85000: 1, 80000: 1, 75000: 1, 70000: 1, 65000: 1, 60000: 1, 55000: 1, 50000: 1, 45000: 1, 40000: 1, 35000: 1, 30000: 1, 25000: 1, 20000: 1, 15000: 1, 10000: 1, 9000: 1, 8500: 1, 8000: 1, 7500: 1, 7000: 1, 6500: 1, 6000: 1, 5500: 1, 5000: 1, 4500: 1, 4000: 1, 3500: 1, 3000: 1, 2500: 1, 2000: 1, 1500: 1, 1000: 1, 950: 1, 900: 1, 850: 1, 800: 1, 750: 1, 700: 1, 650: 1, 600:1, 550: 1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 100:1, 90: 1, 80: 1, 70:1, 60: 1, 50:1, 40:1, 30:1, 25: 1, 20:1, 18:1, 16:1, 15:1, 14:1, 12:1, 10:1, 8:1, 6:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:15, 1:16, 1:18, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, 1:5500, 1:6000, 1:6500, 1:7000, 1: 7500, 1:8000, 1:8500, 1:9000, 1:9500, 1:10000, 1: 15000, 1: 20000, 1:25000, 1: 30000, 1: 35000, 1: 40000, 1: 45000, 1:50000, 1: 55000, 1:60000, 1:65000, 1:70000, 1: 75000, 1:80000, 1:85 000, 1:90000, 1:950000, 1: 100000).

In some embodiments, a ratio of inoculation of the first bacterial strain to the second bacterial strain is between 100000:1 and 1: 100000 (e.g., 100000: 1, 95000: 1, 90000: 1, 85000: 1, 80000: 1, 75000: 1, 70000: 1, 65000: 1, 60000: 1, 55000: 1, 50000: 1, 45000: 1, 40000: 1, 35000: 1, 30000: 1, 25000: 1, 20000: 1, 15000: 1, 10000: 1, 9000: 1, 8500: 1, 8000: 1, 7500: 1, 7000: 1, 6500: 1, 6000: 1, 5500: 1, 5000: 1, 4500: 1, 4000: 1, 3500: 1, 3000: 1, 2500: 1, 2000: 1, 1500: 1, 1000: 1, 950: 1, 900: 1, 850: 1, 800: 1, 750: 1, 700: 1, 650: 1, 600:1, 550: 1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 100:1, 90: 1, 80: 1, 70:1, 60: 1, 50:1, 40:1, 30:1, 25: 1, 20:1, 18:1, 16:1, 15:1, 14:1, 12:1, 10:1, 8:1, 6:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:15, 1:16, 1:18, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, 1:5500, 1:6000, 1:6500, 1:7000, 1: 7500, 1:8000, 1:8500, 1:9000, 1:9500, 1:10000, 1: 15000, 1: 20000, 1:25000, 1: 30 000, 1: 35000, 1: 40000, 1: 45000, 1:50000, 1: 55000, 1:60000, 1:65000, 1:70 000, 1: 75000, 1:80000, 1:85000, 1:90000, 1:950000, 1: 100000).

In some embodiments, a cycle of lysis of any of the bacterial strains described herein can be between 1 hour to 35 days (e.g., 1 hour to 30 days, 1 hour to 28 days, 1 hour to 26 days, 1 hour to 25 days, 1 hour to 24 days, 1 hour to 22 days, 1 hour to 20 days, 1 hour to 18 days, 1 hour to 16 days, 1 hour to 14 days, 1 hour to 12 days, 1 hour to 10 days, 1 hour to 8 days, 1 hour to 7 days, 1 hour to 6 days, 1 hour to 5 days, 1 hour to 4 days, 1 hour to 72 hours, 1 hour to 70 hours, 1 hour to 68 hours, 1 hour to 66 hours 1 hour to 64 hours, 1 hour to 62 hours, 1 hour to 60 hours, 1 hour to 58 hours, 1 hour to 56 hours, 1 hour to 54 hours, 1 hour to 52 hours, 1 hour to 50 hours, 1 hour to 48 hours, 1 hour to 46 hours, 1 hour to 44 hours, 1 hour to 40 hours, 1 hour to 38 hours, 1 hour to 36 hours, 1 hour to 34 hours, 1 hour to 32 hours, 1 hour to 30 hours, 1 hour to 28 hours, 1 hour to 26 hours, 1 hour to 24 hours, 1 hour to 22 hours, 1 hour to 20 hours, 1 hour to 18 hours, 1 hour to 16 hours, 1 hour to 14 hours, 1 hour to 12 hours, 1 hour to 10 hours, 1 hour to 8 hours, 1 hour to 6 hours, 1 hour to 4 hours, 1 hour to 2 hours, 2 hours to 35 days, 2 hours to 30 days, 2 hours to 28 days, 2 hours to 26 days, 2 hours to 25 days, 2 hours to 24 days, 2 hours to 22 days, 2 hours to 20 days, 2 hours to 18 days, 2 hours to 16 days, 2 hours to 14 days, 2 hours to 12 days, 2 hours to 10 days, 2 hours to 8 days, 2 hours to 7 days, 2 hours to 6 days, 2 hours to 5 days, 2 hours to 4 days, 2 hours to 72 hours, 2 hours to 70 hours, 2 hours to 68 hours, 2 hours to 66 hours 2 hours to 64 hours, 2 hours to 62 hours, 2 hours to 60 hours, 2 hours to 58 hours, 2 hours to 56 hours, 2 hours to 54 hours, 2 hours to 52 hours, 2 hours to 50 hours, 2 hours to 48 hours, 2 hours to 46 hours, 2 hours to 44 hours, 2 hours to 40 hours, 2 hours to 38 hours, 2 hours to 36 hours, 2 hours to 34 hours, 2 hours to 32 hours, 2 hours to 30 hours, 2 hours to 28 hours, 2 hours to 26 hours, 2 hours to 24 hours, 2 hours to 22 hours, 2 hours to 20 hours, 2 hours to 18 hours, 2 hours to 16 hours, 2 hours to 14 hours, 2 hours to 12 hours, 2 hours to 10 hours, 2 hours to 8 hours, 2 hours to 6 hours, 2 hours to 4 hours, 4 hours to 35 days, 4 hours to 30 days, 4 hours to 28 days, 4 hours to 26 days, 4 hours to 25 days, 4 hours to 24 days, 4 hours to 22 days, 4 hours to 20 days, 4 hours to 18 days, 4 hours to 16 days, 4 hours to 14 days, 4 hours to 12 days, 4 hours to 10 days, 4 hours to 8 days, 4 hours to 7 days, 4 hours to 6 days, 4 hours to 5 days, 4 hours to 4 days, 4 hours to 74 hours, 4 hours to 70 hours, 4 hours to 68 hours, 4 hours to 66 hours 4 hours to 64 hours, 4 hours to 64 hours, 4 hours to 60 hours, 4 hours to 58 hours, 4 hours to 56 hours, 4 hours to 54 hours, 4 hours to 54 hours, 4 hours to 50 hours, 4 hours to 48 hours, 4 hours to 46 hours, 4 hours to 44 hours, 4 hours to 40 hours, 4 hours to 38 hours, 4 hours to 36 hours, 4 hours to 34 hours, 4 hours to 34 hours, 4 hours to 30 hours, 4 hours to 28 hours, 4 hours to 26 hours, 4 hours to 24 hours, 4 hours to 24 hours, 4 hours to 20 hours, 4 hours to 18 hours, 4 hours to 16 hours, 4 hours to 14 hours, 4 hours to 14 hours, 4 hours to 10 hours, 4 hours to 8 hours, 4 hours to 6 hours, 6 hours to 35 days, 6 hours to 30 days, 6 hours to 28 days, 6 hours to 26 days, 6 hours to 25 days, 6 hours to 24 days, 6 hours to 22 days, 6 hours to 20 days, 6 hours to 18 days, 6 hours to 16 days, 6 hours to 14 days, 6 hours to 12 days, 6 hours to 10 days, 6 hours to 8 days, 6 hours to 7 days, 6 hours to 6 days, 6 hours to 5 days, 6 hours to 4 days, 6 hours to 76 hours, 6 hours to 70 hours, 6 hours to 68 hours, 6 hours to 66 hours 6 hours to 64 hours, 6 hours to 66 hours, 6 hours to 60 hours, 6 hours to 58 hours, 6 hours to 56 hours, 6 hours to 54 hours, 6 hours to 56 hours, 6 hours to 50 hours, 6 hours to 48 hours, 6 hours to 46 hours, 6 hours to 44 hours, 6 hours to 40 hours, 6 hours to 38 hours, 6 hours to 36 hours, 6 hours to 34 hours, 6 hours to 36 hours, 6 hours to 30 hours, 6 hours to 28 hours, 6 hours to 26 hours, 6 hours to 24 hours, 6 hours to 26 hours, 6 hours to 20 hours, 6 hours to 18 hours, 6 hours to 16 hours, 6 hours to 14 hours, 6 hours to 16 hours, 6 hours to 10 hours, 6 hours to 8 hours, 12 hours to 35 days, 12 hours to 30 days, 12 hours to 28 days, 12 hours to 26 days, 12 hours to 25 days, 12 hours to 24 days, 12 hours to 22 days, 12 hours to 20 days, 12 hours to 18 days, 12 hours to 16 days, 12 hours to 14 days, 12 hours to 12 days, 12 hours to 10 days, 12 hours to 8 days, 12 hours to 7 days, 12 hours to 6 days, 12 hours to 5 days, 12 hours to 4 days, 12 hours to 72 hours, 12 hours to 70 hours, 12 hours to 68 hours, 12 hours to 66 hours 12 hours to 64 hours, 12 hours to 62 hours, 12 hours to 60 hours, 12 hours to 58 hours, 12 hours to 56 hours, 12 hours to 54 hours, 12 hours to 512 hours, 12 hours to 50 hours, 12 hours to 48 hours, 12 hours to 46 hours, 12 hours to 44 hours, 12 hours to 40 hours, 12 hours to 38 hours, 12 hours to 36 hours, 12 hours to 34 hours, 12 hours to 312 hours, 12 hours to 30 hours, 12 hours to 28 hours, 12 hours to 26 hours, 12 hours to 24 hours, 12 hours to 22 hours, 12 hours to 20 hours, 12 hours to 18 hours, 12 hours to 16 hours, 12 hours to 14 hours, 1 day to 35 days, 1 day to 30 days, 1 day to 28 days, 1 day to 26 days, 1 day to 25 days, 1 day to 24 days, 1 day to 22 days, 1 day to 20 days, 1 day to 18 days, 1 day to 16 days, 1 day to 14 days, 1 day to 12 days, 1 day to 10 days, 1 day to 8 days, 1 day to 6 days, 1 day to 5 days, 1 day to 4 days, 1 day to 3 days, 1 day to 2 days, 2 days to 35 days, 2 days to 30 days, 2 days to 28 days, 2 days to 26 days, 2 days to 25 days, 2 days to 24 days, 2 days to 22 days, 2 days to 20 days, 2 days to 18 days, 2 days to 16 days, 2 days to 15 days, 2 days to 14 days, 2 days to 12 days, 2 days to 10 days, 2 days to 8 days, 2 days to 6 days, 2 days to 4 days, 2 days to 3 days, 4 days to 35 days, 4 days to 30 days, 4 days to 28 days, 4 days to 26 days, 4 days to 25 days, 4 days to 24 days, 4 days to 22 days, 4 days to 20 days, 4 days to 18 days, 4 days to 16 days, 4 days to 15 days, 4 days to 14 days, 4 days to 12 days, 4 days to 10 days, 4 days to 8 days, 4 days to 6 days, 7 days to 35 days, 7 days to 30 days, 7 days to 28 days, 7 days to 26 days, 7 days to 25 days, 7 days to 24 days, 7 days to 22 days, 7 days to 20 days, 7 days to 18 days, 7 days to 16 days, 7 days to 15 days, 7 days to 14 days, 7 days to 12 days, 7 days to 10 days, 7 days to 8 days, 14 days to 35 days, 14 days to 30 days, 14 days to 28 days, 14 days to 26 days, 14 days to 25 days, 14 days to 24 days, 14 days to 22 days, 14 days to 20 days, 14 days to 18 days, 14 days to 16 days, 14 days to 15 days, 21 days to 35 days, 21 days to 30 days, 21 days to 28 days, 21 days to 26 days, 21 days to 25 days, 21 days to 24 days, 21 days to 22 days, 28 days to 35 days, or 28 days to 30 days; 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days, 22 days, 24 days, 25 days, 26 days, 28 days, 30 days, 32 days, 34 days or 35 days).

The length of a cycle can be regulated by using strains that lyse at different optical density (ODs). Cell lysis can also be regulated by tuning the internal circuitry of the quorum sensing components, e.g., tuning of AHL degradation, tuning lysis of protein degradation, tuning of promoters to increase or decrease expression of molecules involved in the quorum sensing circuitry.

Various methods known in the art can be used to determine whether the quorum threshold is reached. For example, the quorum threshold can be measured using traditional protein quantification methods to measure the level of AHL expression in the culture medium. The quorum threshold can also be measured using reporter proteins driven by the luxI promoter. In some embodiments, the reporter protein is a fluorescent protein, a bioluminescent luciferase reporter, a secreted blood/serum or urine reporter (e.g., secreted alkaline phosphatase, soluble peptides, Gaussian luciferase).

Various methods are known in the art to determine and/or measure cell lysis. For example, cell lysis can be determined phenotypically using microscopy by the change in intensity of transmitted light and/or absorbance at various wavelengths including 600 nm light. In some embodiments, bacterial cell lysis is synchronized. In other embodiments, bacterial cell lysis is not synchronized. Synchronized lysis can be measured via optical density at 600 nm absorbance (OD600) in a plate reader or other quantitative instruments.

Systems

Provided herein are systems that include a culture of a bacterial strain, wherein the bacterial strain comprises: a first nucleic acid sequence encoding a first activator polypeptide (e.g., any of the first activator polypeptides described herein), wherein expression of the first activator polypeptide results in production of a quorum sensing molecule precursor, wherein the first nucleic acid sequence is operably linked to a promoter; a second nucleic acid sequence encoding a second activator polypeptide (e.g., any of the second activator polypeptides described herein), wherein expression of the second activator polypeptide results in production of a quorum sensing molecule, wherein the second nucleic acid sequence is operably linked to a first activatable promoter; a third nucleic acid sequence encoding a third activator polypeptide (e.g., any of the third activator polypeptides described herein), wherein expression of the third activator polypeptide results in activation of the quorum sensing system, wherein the third nucleic acid sequence is operably linked to a second activatable promoter; and a fourth nucleic acid sequence encoding a gene of interest (e.g., any of the genes of interest described herein), wherein the fourth nucleic acid sequence is operably linked to a third activatable promoter; and an inducer molecule; and a quorum sensing molecule that enables induction of quorum sensing.

Provided herein are systems that can include a co-culture of at least two bacterial strains (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), wherein the at least two bacterial strains can include a first bacterial strain having at least a portion of a first synchronized lysis circuit, wherein the first synchronized lysis circuit comprises a first lysis polypeptide and a first activator polypeptide and wherein the first lysis polypeptide is activated by the first activator polypeptide.

In some embodiments described herein, a system comprises a co-culture of at least a first bacterial strain and a second bacterial strain, wherein the first bacterial strain has at least a portion of a first synchronized lysis circuit, wherein the first synchronized lysis circuit comprises a first lysis polypeptide, a first activator polypeptide, and a first polypeptide stabilizing element, and wherein the first lysis polypeptide is activated by the first activator polypeptide, and wherein the second bacterial strain has at least a portion of a second synchronized lysis circuit, wherein the second synchronized lysis circuit comprises a second lysis polypeptide, a second activator polypeptide, and a second polypeptide stabilizing element, and wherein the second lysis polypeptide is activated by the second activator polypeptide, and wherein the first and second synchronized lysis circuits are orthogonal in that each has no or substantially no effect upon the other. As used herein, “substantially no effect” means no measurable effect on the activatable promoter, as measured by the expression of the activatable promotor of a fluorescent protein. In some aspects, the first bacterial strain can include the first lysis polypeptide. In some aspects of any of the systems described herein, the first bacterial strain can include the first activator polypeptide.

In some aspects of any of the systems described herein, the at least two bacterial strains further can include a second bacterial strain.

In some aspects of any of the systems described herein, the second bacterial strain can include the first activator polypeptide.

In some aspects of any of the systems described herein, each of the first bacterial strain and the second bacterial strain can include the first activator polypeptide.

In some aspects of any of the systems described herein, the first lysis polypeptide of the first bacterial strain operates independent of at least one other bacterial strain in the co-culture. In some embodiments, the first lysis polypeptide of the first bacterial strain operates independent of at least the second bacterial strain. In some embodiments, the first lysis polypeptide of the first bacterial strain operates independent of at least one bacterial strain in the system that is not the second bacterial strain.

In some aspects of any of the systems described herein, the first lysis polypeptide of the first bacterial strain responds to a signal generated by at least one other bacterial strain in the co-culture. In some embodiments, the first lysis polypeptide of the first bacterial strain responds to a signal generated by at least the second bacterial strain. In some embodiments, the first lysis polypeptide of the first bacterial strain responds to a signal generated by at least one bacterial strain in the system that is not the second bacterial strain.

In some aspects of any of the systems described herein, the signal is a quorum sensing signal. In some aspects of any of the systems described herein, the first activator polypeptide encodes a quorum sensing signal. In some aspects of any of the systems described herein, the second activator polypeptide encodes a quorum sensing signal. In some embodiments, the quorum sensing signal can be a quorum sensing signaling molecule. In some embodiments, one or more of the bacterial strains respond to a quorum sensing signal. In some embodiments, the quorum sensing signals for two or more of the bacterial strains are different quorum sensing signals. In some embodiments, the quorum sensing signals for two or more of the bacterial strains are the same quorum sensing signals.

In some embodiments, the quorum sensing signaling molecule for the first and second synchronized lysis circuits are orthogonal in that each has no measurable effect upon the other.

In some aspects of any of the systems described herein, the second bacterial strain has at least a portion of a second synchronized lysis circuit, wherein the second synchronized lysis circuit comprises a second lysis polypeptide and a second activator polypeptide.

In some aspects of any of the systems described herein, the second bacterial strain comprises the second lysis polypeptide.

In some aspects of any of the systems described herein, second bacterial strain comprises the second activator polypeptide.

In some aspects of any of the systems described herein, the first bacterial strain comprises the second activator polypeptide.

In some aspects of any of the systems described herein, the second lysis polypeptide of the second bacterial strain operates independent of at least the first bacterial strain.

In some aspects of any of the systems described herein, the second lysis polypeptide of the second bacterial strain responds to a signal generated by the first bacterial strain.

In some aspects of any of the systems described herein, at least one of the at least two bacterial strains (e.g., at least one of a first bacterial strain and a second bacterial strain) has a growth advantage compared to at least one other bacterial strain. In some embodiments, at least the first bacterial strain has a growth advantage compared to at least the second bacterial strain. In some embodiments, at least the second bacterial strain has a growth advantage compared to at least the first bacterial strain. In some embodiments, at least the first bacterial strain has a growth advantage compared to a bacterial strain present in the system that is not the second bacterial strain. In some embodiments, at least the second bacterial strain has a growth advantage compared to a bacterial strain present in the system that is not the first bacterial strain. In some embodiments, the system can contain multiple orthogonal co-lysis circuits. For example, a system described herein could include a first co-lysis circuit comprising a first bacterial strain and a second bacterial strain as described herein, as well as a second co-lysis circuit comprising a third bacterial strain and a fourth bacterial strain. In some embodiments, the third and fourth bacterial strains each comprise a lysis polypeptide having a lysis gene under the control of an activatable promoter; and an activator polypeptide having an activator gene, the expression of which promotes the accumulation of a quorum sensing molecule, wherein both the activatable promoter of the lysis gene and the expression of the activator gene is activated by the quorum sensing molecule, wherein the quorum-sensing molecule of the third strain is different from the quorum-sensing molecule of the fourth strain, and wherein each quorum-sensing molecule of the third and fourth strains has no or substantially no effect on the activatable promoter of the lysis gene of the other strain. In some embodiments, the third and fourth bacterial strains can be described in the same manner that the first and second bacterial strains have been described herein. In some embodiments, a system described herein can contain 3, 4, 5, 6, 7, 8, 9, 10, or more co-lysis circuits.

In some aspects of any of the systems described herein, the first bacterial strain is competitive with at least one other bacterial strain in the co-culture. In some embodiments, the first bacterial strain is competitive with at least the second bacterial strain in the co-culture. In some embodiments, the first bacterial strain is competitive with at least one other bacterial strain in the co-culture that is not the second bacterial strain.

In some aspects of any of the systems described herein, the co-culture is stable for at least 48 hours.

In some aspects of any of the systems described herein, the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) do not comprise engineered positive or negative interactions between each other.

In some aspects of any of the systems described herein, at least one of the at least two bacterial strains (e.g., at least one of a first bacterial strain and a second bacterial strain) dynamically controls its population without exogenous input.

In some aspects of any of the systems described herein, each of at least two of the at least two bacterial strains (e.g., each of at least a first bacterial strain and a second bacterial strain) dynamically controls its own population without exogenous input.

In some aspects of any of the systems described herein, the system can further include one or more polypeptide stabilizing elements. In some aspects of any of the systems described herein, the polypeptide stabilizing element is selected from a toxin/antitoxin system and an actin-like protein partitioning system.

In some aspects of any of the systems described herein, the first activator polypeptide encodes a degradation tagging sequence.

In some aspects of any of the systems described herein, the second activator polypeptide encodes a degradation tagging sequence.

In some aspects of any of the systems described herein, the first activator polypeptide encodes an N-acyl homoserine lactone.

Compositions and Kits

Also provided herein are compositions (e.g., pharmaceutical compositions) that include any of the bacterial strains described herein (e.g., any of the bacterial strains including any on the nucleic acid sequences described herein). Any of the pharmaceutical compositions can include any of the bacterial strains described herein and one or more (e.g., 1, 2, 3, 4, or 5) pharmaceutically or physiologically acceptable carriers, diluents, or excipients. In some embodiments, any of the pharmaceutical compositions described herein can include one or more buffers (e.g., a neutral-buffered saline, a phosphate-buffered saline (PBS)), one or more carbohydrates (e.g., glucose, mannose, sucrose, dextran, or mannitol), one or more proteins, polypeptides, or amino acids (e.g., glycine), one or more antioxidants, one or more chelating agents (e.g., glutathione or EDTA), one or more preservatives, and/or a pharmaceutically acceptable carrier (e.g., PBS, saline, or bacteriostatic water).

Also provided are kits that include any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, a kit can include a solid composition (e.g., a lyophilized composition including any of the bacterial strains described herein) and a liquid for solubilizing the lyophilized composition.

In some embodiments, the kit includes a vial including any of the pharmaceutical compositions described herein (e.g., formulated as an aqueous pharmaceutical composition).

In some embodiments, the kit can include instructions for performing any of the methods described herein.

Methods of Controlling Population Levels

Also provided herein are methods for controlling population density using any of the inducible quorum sensing systems described herein. In some embodiments, the concentration of p-coumaric acid (pCA) added to induce quorum sensing results in sustained population density. In some embodiments, the concentrations of pCA added to induce quorum sensing results in oscillation of population density. In some embodiments, the inducible quorum sensing system includes activation of a lysis gene, where activation of the lysis gene results in reduction in one or more of the bacterial strains.

In some embodiments, the method for controlling population density using any of the inducible quorum sensing systems includes contacting the one or more bacterial strains with the inducer molecule (e.g., pCA) and reducing the level of one or more of the bacterial strains by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of the total population of bacteria in the culture as compared to a bacterial culture including one or more of the bacterial strains described herein not contacted with the inducer molecule (e.g., pCA).

Also provided herein are methods for controlling cargo release using any of the inducible quorum sensing systems described herein. In some embodiments, the inducible quorum sensing includes activation of a lysis gene, wherein activation of the lysis gene results in controlled release of cargo. In some embodiments, controlled release of cargo can control population density.

Methods of Treatment

Also provided herein are methods for treating a disease by administering to the subject a therapeutically effective amount of any of the bacterial strains described herein, or any of the pharmaceutical compositions described herein. Additional non-limiting aspects of the bacterial strains, pharmaceutical compositions, kits, and methods are described herein and can be used in any combination without limitation.

In some embodiments of any of the methods described herein, the subject has a cancer or an infection. In some embodiments wherein the subject has a cancer, the cancer can be, e.g., a primary tumor, or a metastatic tumor. In some embodiments, the cancer is a non-T-cell-infiltrating tumor. In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of: glioblastoma, squamous cell carcinoma, breast cancer, colon cancer, hepatocellular cancer, melanoma, neuroblastoma, pancreatic cancer, and prostate cancer. Treatment of multiple cancer types at the same time is contemplated by and within the present disclosure.

In some instances, the subject having the cancer may have previously received cancer treatment (e.g., any of the cancer treatments described herein).

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the bacterial strains of the present disclosure and practice the claimed methods. The following working examples specifically point out various aspects of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

Examples

Example 1. Design and Characterization of the Inducible Quorum Sensing Sytem (iQS)

To design an exemplary iQS system, the two-step pathway which converts pCA into p-coumaroyl-HSL (pC-HSL) was genetically reconstructed through the production of the intermediate molecule p-coumaroyl-CoA (FIG. 1A). The first conversion is catalyzed by the p-coumaric acid-CoA ligase encoded by the 4CL2nt gene the plant Nicotiana tabacum while the second step is catalyzed by the RpaI synthase. Additionally, superfolder green fluorescent protein (sfGFP) was added to be able to monitor the inducible dynamics. In some cases, all genes, with the exception of 4CL2nt, are driven by the luxI quorum sensing promoter, which has been shown to perform better than the native promoter of R. palustris when heterologously expressed. Escherichia Coli was chosen as the host chassis for the double plasmid circuit. The methods described in Example 1 are also applicable to Examples 2, 3, and 4.

Plasmids and Strains

The iSLC and SLC strains were both cultured in lysogeny broth (LB) media with 50 μg mL⁻¹ kanamycin, 34 μg mL⁻¹ chloramphenicol and 0.2% glucose for strains containing ColE1 origin and p15A origin plasmids in a 37° C. shaking incubator. Plasmid pTD103 RpaR-RpaI-LAA-sfGFP²¹, ptD103-CFP were constructed by as described in previous studies (see, e.g., FIG. 11). Both plasmids pAM014 and pAM021 were obtained by inserting the 4CL2nt gene under the constitutive promoter J23106 from the Anderson promoter library. The 4CL2nt gene and the promoter were synthesized with overlapping PCR of long oligonucleotides (IDT). All plasmids were constructed by Gibson assembly followed by transformation into DH5α (Thermofisher) chemically competent E. coli. All plasmids were verified by Sanger sequencing before transformation into E. coli strain MG1655.

Microfluidics and Microscopy

The microscopy and microfluidics techniques used are similar to those previously reported. The microfluidic devices were constructed from PDMS (polydimethylsiloxane), which was molded and baked on a silicon wafer with micronscale features formed by crosslinked photoresist. Once the PDMS hardened, it was peeled off, and individual devices were cut out. In order to connect fluid lines to the device, holes where punched in correspondence of the inlets and outlets of the device. Afterwards, the devices were bonded onto glass slides using plasma activation. Before each experiment, the devices were left for 30 minutes in a vacuum chamber. Meanwhile, 1 mL of overnight cell culture was spun down by centrifugation and re-suspended in 10 μL of fresh media with appropriate antibiotics. After taking the device out of the vacuum chamber, a single droplet of resuspended cells was positioned in correspondence of the outlet opening. Similarly, droplets of sterile fresh media were placed in correspondence of the inlets openings. In all cases, 0.075% Tween20 was added to the medium to prevent cells from sticking to the PDMS walls. After all chip features were wetted, the fluids lines were plugged in and the height of the inlet was raised 10 to 40 cm above the device. The outlet syringe was instead placed at the same height of the device. For co-culturing experiments in FIG. 4, cells were cultured individually overnight and eventually spun down and re-suspended together (1:1 ratio) allowing for a single droplet to be loaded on the device. All experiments shown in FIG. 2 and FIG. 4 were performed in a sidetrap array device with bacteria growth chambers approximately 100×80 μm in area and approximately 1.2 μm in height. The upstream channels consists of a series of dividing serpentine which allow for sequential dilutions of the two input media, generating a gradient of eight different inducer concentrations. For the kill switch experiments, reported in FIG. 3, a simpler device was used with a single input and an ordered array of traps. The dimensions of the traps are the same as the ones described for the gradient device. Experiments in FIG. 2 and FIG. 4 where carried out by connecting a syringe with LB+antibiotics+0.075% Tween20 as inlet 1 and a syringe with LB+antibiotics+0.075% Tween20+1 μM of p-coumaric acid as inlet 2. P-coumaric acid inductions for microfluidic experiments in FIG. 3B were performed by unplugging the syringe with pure media and substituting it with a second syringe containing media plus the appropriate p-coumaric acid concentration. For microscopy, the same system was used as described in previous studies. In brief, images were acquired with a Nikon TI2 using a Photometrics CoolSnap cooled CCD camera. The scope and accessories were programmed using the Nikon Elements software. The microscope was housed in a plexiglass incubation chamber maintained at 37° C. by a heating unit. Phase-contrast images were taken at 4× and 10× magnification at 50-100 μs exposure times. At 4× magnification fluorescence exposure times were 2 seconds at 30% intensity for both gfp and cfp while at 10× magnification they were 200 μs at 30% intensity for both GFP and CFP. Images were taken every 6 minutes for each experiment. For induction experiments, imaging was paused while syringes were swapped.

Data Analysis

Fluorescence intensity profiles were obtained by analyzing frames from the fluorescent channels. The mean fluorescence values were calculated by drawing a rectangle surrounding each trap individually and extracting the z-axis profile on ImageJ. Fluorescence values shown in FIG. 2 were normalized by dividing all data by a constant factor. In addition, the subset of traps for each column was normalized by subtracting the minimum value among the traps in the subset. Transmitted light data from FIG. 3B was normalized by subtracting the minimum value for each time trace. Fluorescence data shown in FIG. 4 was normalized by first dividing the subsets of traps by their overall maximum values and subsequently subtracting the respective minimum values. In addition, the data in FIG. 4 was smoothed with the command smoothdata( ) in Matlab. Heatmaps were generated in Matlab using the function heatmap( ). For the co-culture experiments, when overlap between the gfp and cfp channels was observed, values were corrected taking into consideration the image frames in order to subtract overlapping signal.

Plate Reader Experiments

For plate reader experiments, the appropriate strains were seeded from a −80° C. glycerol stock into 3 ml LB with 0.2% glucose and appropriate antibiotics and incubated in a 37° C. shaking incubator. The following day, 2 μL of overnight culture were added to 200 μL fresh media containing appropriate antibiotics in a standard Falcon tissue culture 96-well flat bottom plate. Cells were incubated at 37° C. shaking in a Tecan Infinite M200 Pro. Cells were grown for about 12 hours. The optical density at 600 nm absorbance was measured every 10 minutes.

Cells Survival Assay

Cell viability assays to test the efficacy of iQS as a kill switch were done by measuring colony forming units (CFUs), following a protocol found in the literature. Cells were grown under survival conditions in LB with 0.2% glucose which inhibits the LuxI promoter thanks to the presence of a binding site for the CAP-cAMP activating complex. Following overnight growth, they were transferred into four cultures of fresh LB medium with 0.2% glucose, 300 nM p-coumaric acid, 500 nM p-coumaric acid and 1 μM p-coumaric acid, respectively. Samples were collected every 2 hours and serially diluted in PBS over a 7-log range and spotted (2 μL) onto LB agar plates with 0.2% glucose. The equations used are: CFU/ml=(number of colonies)×(dilution factor)/0.002 mL, survival ratio (log 10)=log(CFU/ml with glucose)/(CFU/ml with p-coumaric acid).

Results

Predictions were formulated that inducibility would expand the range of population dynamics by including an ‘OFF’ state at low inducer concentration and an ‘ON’ state at high inducer concentrations which are both independent of population density. On the other hand, for a range of intermediate concentrations the iQS would behave like a standard QS system by exhibiting the typical population density dependent activation (FIG. 1B). To investigate how the engineered iQS strain would relate to these expected dynamics, microfluidic devices were used, which enabled the simultaneous exposure of varying pCA concentrations to different subgroups of cells, Time-lapse fluorescence microscopy was used to observe fluorescence expression as a function of population size and inducer concentration. The data matched the expected dynamics of an inducible quorum sensing system when exposed to zero, medium (1:5 nM) or high (1 μM) inducer concentrations (FIG. 1C). Additionally, further characterization of the iQS response over a broader range of pCA concentrations was performed using a microwell plate reader. A concentration of zero of the inducer molecule resulted in the circuit being OFF except for a baseline of leaky expression. Intermediate levels (1 nM and 10 nM) induced the typical population-dependent switch-like behavior with a steep increase in fluorescence signal at an optical density threshold of around 0.3. Finally, high concentrations (100 nM to 10 μM) resulted in a linear relationship between fluorescence expression and population density, confirming the assumption of a population independent ‘ON’ state (FIG. 10). As a control, the same experiment was repeated with a wild type E. Coli strain and observed that the growth curves were unaffected, confirming pCA is not toxic within the characterized range (FIG. 1E).

Example 2. Characterization of the Inducible Synchronized Lysis Circuit (iSLC)

To demonstrate the usefulness of the iQS over non-inducible quorum sensing based circuits, the iQS was coupled with the expression of a lysis gene to create a platform for inducible population dependent bacterial cargo delivery (FIG. 2A). To qualitatively predict the dynamics of this iSLC strain (induced synchronized lysis circuit), a deterministic model was developed as described below, which is also applicable to Examples 1, 3, and 4.

Modeling

A deterministic model was constructed to qualitatively describe the dynamic behavior of the iSLC strain. The model is based on a set of six ODEs (orindary differential equation) which track the evolution of the following six variables: the cell number into a single microfluidic trap (N), the external concentration of p-coumaroyl-HSL (pH), the intracellular concentration of lysis protein (L), the intracellular concentration of the variable RpaI (I), the intracellular concentration of 4CL2nt enzyme (E) and the intracellular concentration of the intermediate compound p-coumaroyl-CoA (pA). The inducer concentration is kept fixed for each simulation and it is defined together with the initial conditions.

A non-zero p-coumaric acid concentration induces the production of the intermediate molecule p-coumaroyl-CoA (pA) through the enzyme 4CL2nt (E). The production term of the latter is a constant variable due to the constitutive promoter which drives it. The intermediate product (pA) is transformed into the quorum sensing molecule pC-HSL through the RpaI enzyme(I). Once a threshold value of extracellular pC-HSL is reached, the intracellular production of the luxI driven genes (RpaI, RpaR and E) are brought to the ON state, thanks to the positive feedback provided by the QS promoter. Activation of the promoter also leads to cell killing as a consequence of the lysis gene expression. The assumption is that the quorum sensing molecule diffuses quickly through the membrane, therefore there is distinguishing between intracellular and extracellular HSL concentration. In addition, the assumption is made that the RpaR-pC-HSL binding is instantaneous, so that the model can be simplified by ignoring the dynamics of the binding complex. Degradation of all proteins (L, I, E) is associated to dilution due to cell growth (μG) as well as basal intracellular degradation (γL and γI). In addition to those terms, RpaI (I) is also actively degraded by ClpXP proteases (γC). Overall, the model can accurately predict the three main dynamics of the cell population as the inducer concentration is varied. With zero inducer concentration, the population grows reaching a steady state value. At very small inducer concentrations, the population undergoes small amplitude lysis events followed by steady state. A finite range of intermediate inducer values was observed which resulted in sustained oscillations of population density. Finally, total population death was observed with no survivors for high concentrations (FIG. 12). Furthermore, visualization of the non-linear dynamics of the system was performed using phase portraits obtained by plotting N (cell number) against L (lysis protein) (FIG. 13). As the p-coumaric acid concentration is increased, the simulations show a first transition from a stable spiral to a limit cycle which indicates sustained oscillations. A further increase in the inducer parameter causes the limit cycle to disappear in favor of a stable fixed point. All plots are generated in MATLAB.

$\begin{array}{cc}\frac{\mathrm{dN}}{\mathrm{dt}}=\text{?}*N*\left(N_0-N\right)-N*\frac{k*L^n}{{\left(L_0\right)}^n+L^n}& \left(1\right)\end{array}$ $\begin{array}{cc}\frac{d\mathrm{pA}}{\mathrm{dt}}=\text{?}*\mathrm{inducer}*E-\text{?}*\mathrm{pA}-\text{?}*\mathrm{pA}& \left(2\right)\end{array}$ $\begin{array}{cc}\frac{d\mathrm{pH}}{\mathrm{dt}}={\mu }_H*N*I*\mathrm{pA}-\frac{u*\mathrm{pH}}{1+\text{?}}& \left(3\right)\end{array}$ $\begin{array}{cc}\frac{dL}{\mathrm{dt}}=C_i*\left({\alpha }_0+\frac{{\alpha }_H*\text{?}}{1+\text{?}}\right)-{\gamma }_L*L-\text{?}*L& \left(4\right)\end{array}$ $\begin{array}{cc}\frac{\mathrm{dI}}{\mathrm{dt}}=C_i*\left({\alpha }_0+\frac{{\alpha }_H*\text{?}}{1+\text{?}}\right)-{\gamma }_I*I-{\mu }_G*I& \left(5\right)\end{array}$ $\begin{array}{cc}\frac{\mathrm{dE}}{\mathrm{dt}}=C_i*\text{?}-{\gamma }_4*E-\text{?}*E-\text{?}*E& \left(6\right)\end{array}$ $\text{?}\text{indicates text missing or illegible when filed}$

Model parameters were based on a similar model previously published. Compared to the previously used lux quorum sensing system, the iSLC showed higher promoter leakiness which was taken into account by increasing the basal production term (α0). The parameter values used in the model are μG=0.2 (dilution due to cell growth), N0=20 (cell capacity of a single trap), k=10 (maximum rate of cell lysis), L0=1 (concentration of lysis protein resulting in half maximum lysis), n=2 (Hill's coefficient), Cl=0.5 (copy number of the lysis gene), Ci=1 (RpaI gene copy number), Pconst=20 (strength constitutive promoter driving gene 4CL2nt), γ4=2 (degradation of enzyme 4CL2nt), α0=1 (pLux basal leakiness), αH=20 (Lux promoter RpaR-pC-HSL induced production), H0=4.5 (pC-HSL-RpaR binding affinity to pLux), γL=2 (lysis protein basal degradation), γI=2 (RpaI protein basal degradation), γC=12 (paI protein degradation due to ClpXP), γCoA=2 (p-coumaroyl-CoA basal degradation), μ4=30 (con-version rate of p-coumaric acid into p-coumaroyl-CoA), μH=15 (pC-HSL production rate) and μ=12 (maximum AHL clearance rate due to flow).

Results

A bifurcation plot was obtained by simulating the steady state values of cell population as a function of pCA concentration, predicting the emergence of three main population regimes FIG. 2B). Small amplitude lysis events followed by steady growth are predicted at low inducer concentrations. Intermediate pCA concentrations result in sustained oscillations in population density. Finally, high inducer concentrations lead to a single lysis event with zero survivors. Therefore, the iSLC was expected to considerably expand the range of possible population dynamics by adding a quiescent state of circuit inactivation and a termination state in which all cells undergo lysis, regardless of population density (FIG. 2C).

A preliminary test using a microwell plate reader showed an inverse correlation between the population OD (optical density) at lysis and the inducer concentration (FIG. 5). Interestingly, the presence of a lysis event at zero pCA concentration proved to be caused by leaky expression of the Lexi promoter in the sole presence of RpaR (FIGS. 6A-6B). To fully visualize the dynamics of the iSLC strain over time, microfluidic devices were used with an upstream serpentine of branching channels to generate a gradient of eight different inducer concentrations (FIG. 2D and FIG. 7).

Using fluorescence microscopy to monitor population dynamics, the same three emerging population behaviors predicted by the mathematical model were observed (FIG. 2E and FIG. 2F). With zero inducer, constant cell growth was observed with sporadic asynchronous lysis due to promoter leakiness. Intermediate inducer concentrations (15 nM) resulted in sustained synchronized oscillations with an average period of approximately 200 minutes. Finally, high pCA concentrations (110 nM to 1 WV) caused universal cell lysis regardless of population density, with only a small fraction (<3% of all microfluidics traps) able to survive (FIG. 8).

Example 3. Characterization of the iSLC Kill Switch Properties in Microfluidics and Liquid Culture

Experiments were performed to investigate wether inducer concentrations could be modulated during culture to show the culture can switch between states of constant growth (circuit quiescence), synchronized oscillations in population density (cyclic cargo release) and inducible population death (kill switch). To confirm this principle, experiments were performed to test the ability to drive the population dynamics from state one (population growth) to state three (population death) through pCA induction (FIG. 3A). Experiments were performed in microfluidic devices by initially growing the cells with zero inducer concentration (circuit quiescence). Following complete saturation of all traps, induction was performed with 500 nM pCA. A synchronized lysis event was observed throughout the entire device which resulted in cell death in more than 97% of the 406 microfluidic traps present (FIG. 3B and FIG. 3C). To test the reproducibility of this property in larger volumes, the iSLC strain was grown in 3 mL culture tubes with and without inducer for a period of eight hours. In the presence of p-coumaric acid, a several order of magnitude decrease in cell survival was observed which was independent of inducer concentration (FIG. 3D). This ability to bypass population dependency and intentionally decimate the population, regardless of its density, may serve as an integrated kill switch to regulate strain removal in space and time.

Example 4. iQS Enabled Modulation of Orthogonal Multi-Strain Dynamics

Experiments were performed to investigate the orthogonal properties of the iQS circuit by co-culturing the iSLC with a non-inducible SLC strain based on the lux QS system, one of the most commonly used and well understood QS systems in synthetic biology (FIG. 4A). First, the absence of crosstalk between p-coumaric acid and the lux QS system was demonstrated by growing the iSLC strain in a range of different pCA concentrations (FIG. 9). Next, a previously described microfluidic chip was used (FIG. 2D and FIG. 7) to co-culture the iSLC and SLC strains (starting at 1:1 ratio) under a gradient of inducer concentrations. Interestingly, it was observed that by varying pCA concentration, precise control over community composition of the microfluidic traps could be achieved. Without inducer, the quiescent iSLC strain was able to displace the SLC strain due to an advantage in population growth rate. At intermediate concentrations (15 nM), stable co-culture of both strains were maintained with independently sustained oscillations for more than 40 hours. High pCA concentrations (110 nM) resulted in the death of all iSLC cells, allowing the SLC strain to fully take over (FIG. 4B and FIG. 4C). For inducer values higher than 340 nM, the dynamics were unanimously characterized by iSLC death and SLC takeover (FIG. 10). As demonstrated, the absence of cross-talk between the pCA derived signaling molecule and the majority of well-characterized quorum sensing systems, enables the simultaneous use of multiple quorum sensing in co-culture, providing exciting new possibilities for microbial community engineering.

EMBODIMENTS

Embodiment A1. A Method of Inducing Quorum Sensing, the Method Comprising:

(a) culturing a bacterial strain, wherein the bacterial strain comprises

• • (i) a first nucleic acid sequence encoding a first activator polypeptide that is capable of activating a quorum sensing system, wherein the first nucleic acid sequence is operably linked to an activatable promoter;
  • (ii) a second nucleic acid sequence encoding a second activator polypeptide that is capable of activating a quorum sensing system, wherein the second nucleic acid sequence is operably linked to an activatable promoter; and
  • (iii) a third nucleic acid sequence encoding a third activator polypeptide that is capable of activating a quorum sensing system, wherein the third nucleic acid sequence is operably linked to a promoter;

(b) contacting the bacterial strain with an inducer molecule; and

(c) converting the inducer molecule into a quorum sensing molecule, thereby allowing induction of quorum sensing in the bacterial strain.

Embodiment 2. The method of embodiment 1, wherein the quorum sensing system is a Lux-like quorum sensing system selected from the group consisting of: a lux system from Vibrio fischeri; a las system from Pseduomonas aeruginosa; a rhl system from Pseduomonas aeruginosa; a tra system from Agrobacterium tumefaciens; a rpa system from Rhodopseudomonas palustris; an ahy system from Aeromonas hydrophilia; a sma system from Serratia marcescens; cer system from Rhodobacter sphaeroides; and an exp system from Sinorhizobium meliloti.

Embodiment 3. The method of any one of embodiments 1-2, wherein the quorum sensing system is an rpa quorum sensing system.

Embodiment 4. The method of any one of embodiments 1-2, wherein the quorum sensing system is a lux quorum sensing system.

Embodiment 5. The method of any one of embodiments 1-4, wherein the quorum sensing system combines elements of the rpa quorum sensing system and the lux quorum sensing system.

Embodiment 6. The method of any one of embodiments 1-5, wherein the activatable promoter comprises a LuxR-AHL activatable luxI promoter.

Embodiment 7. The method of any one of embodiments 1-5, wherein the activatable promoter comprises an RpaR-AHL activatable RpaI promoter.

Embodiment 8. The method of any one of embodiments 1-7, wherein the first polypeptide comprises a first enzyme, wherein expression of the enzyme produces a quorum sensing molecule precursor.

Embodiment 9. The method of embodiment 8, wherein the second enzyme is a p-coumaric acid-CoA ligase.

Embodiment 10. The method of embodiment 9, wherein the p-coumaric acid-CoA ligase is encoded by the 4CL2nt gene from the plant Nicotiana tabacum.

Embodiment 11. The method of any one of embodiments 1-10, wherein the quorum sensing molecule precursor is p-Coumaroyl-CoA.

Embodiment 12. The method of any one of embodiments 1-11, wherein the second activator polypeptide comprises a second enzyme, wherein expression of the enzyme produces a quorum sensing molecule.

Embodiment 13. The method of embodiment 12, wherein the enzyme is a 4-coumaroyl-homoserine lactone synthase (RpaI) synthase.

Embodiment 14. The method of embodiment 13, wherein the RpaI synthase is encoded by the RpaI gene from the plant Rhodopseudomonas palustris.

Embodiment 15. The method of any one of embodiments 1-14, wherein the quorum sensing molecule is p-Coumaroyl-HSL.

Embodiment 16. The method of any one of embodiments 1-15, wherein the third activator polypeptide comprises a signaling molecule, wherein the signaling molecule interacts with the quorum sensing molecule.

Embodiment 17. The method of embodiment 16, wherein the signaling molecule is a HTH-type quorum sensing-dependent transcriptional regulator (RpaR).

Embodiment 18. The method of embodiment 17, wherein the RpaR signaling molecule is encoded by the RpaR gene from the plant Rhodopseudomonas palustris.

Embodiment 19. The method of any one of embodiments 1-18, wherein the interaction of RpaR and the quorum sensing molecule p-Coumaroyl-HSL results in transcriptional regulation of downstream target genes.

Embodiment 20. The method of any one of embodiments 1-19, wherein the first activator polypeptide is a p-coumaric acid-CoA ligase, the second activator polypeptide is a RpaI synthase, and the third activator polypeptide is a RpaR signaling molecule.

Embodiment 21. The method of any one of embodiments 1-20, wherein the inducer molecule is p-coumaric acid.

Embodiment 22. The method of any one of embodiments 1-21, wherein the first activator polypeptide is p-coumaric acid-CoA ligase that produces p-Coumaroyl-CoA from the substrate p-coumaric acid.

Embodiment 23. The method of any one of embodiments 1-22, wherein the second activator polypeptide is RpaI synthase that produces p-Coumaroyl-HSL from the substrates p-Coumaroyl-CoA.

Embodiment 24. The method of any one of embodiments 1-23, wherein the third activator polypeptide is a RpaR signaling molecule that interacts with the p-Coumaroyl-HSL.

Embodiment 25. The method of any one of embodiments 1-24, wherein the method further comprises a fourth nucleic acid sequence encoding a gene of interest, wherein the fourth nucleic acid sequence is operably linked to a promoter.

Embodiment 26. The method of embodiment 25, wherein the gene of interest encodes a bacteriophage lytic protein capable of forming a lesion in a host cell's membrane.

Embodiment 27. The method of embodiment 26, wherein the lysis gene is from a bacteriophage ΦX174.

Embodiment 28. The method of any one of embodiments 1-27, wherein the method further comprises a fifth nucleic acid sequence encoding a reporter polypeptide selected from a nucleic acid encoding a green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), or a variant thereof.

Embodiment 29. The method of embodiment 28, wherein the fifth nucleic acid sequence is operably linked to a promoter or is operably linked to the first, second, third, and/or fourth nucleic acid sequences.

Embodiment 30. The method of any one of embodiments 1-24, wherein the method comprises a vector comprising the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence.

Embodiment 31. The method of any one of embodiments 1-27, wherein the method comprises a vector comprising the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, and the fourth nucleic acid sequence.

Embodiment 32. The method of any one of embodiments 1-24 or 28-29, wherein the method comprises a vector comprising the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, and the fifth nucleic acid sequence.

Embodiment 33. The method of any one of embodiments 1-29, wherein the method comprises a vector comprising the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, and the fifth nucleic acid sequence.

Embodiment 34. The method of any one of embodiments 1-24, wherein the method comprises a first vector and a second vector, wherein the first vector comprises a first nucleic acid sequence and the second vector comprises the second nucleic acid sequence and the third nucleic acid sequence.

Embodiment 35. The method of any one of embodiments 1-24, wherein the method comprises a first vector, a second vector, and a third vector, wherein (i) the first vector comprises the first nucleic acid sequence, (ii) the second vector comprises the second nucleic acid sequence and (iii) the third vector comprises the fourth nucleic acid sequence.

Embodiment 36. The method of any one of embodiments 1-35, wherein the one or more vectors are integrated into the genome of the bacterial strain.

Embodiment 37. The method of any one of embodiments 30-36, wherein the one or more vectors each comprise a vector-stabilizing element.

Embodiment 38. The method of embodiment 37, wherein the vector-stabilizing element is a toxin/antitoxin system or an actin-like protein partitioning system.

Embodiment 39. The method of any one of embodiments 1-38, wherein the culturing occurs in a microfluidic device.

Embodiment 40. The method of any one of embodiments 1-39, wherein the bacterial strain is E. coli.

Embodiment 41. The method of any one of embodiments 1-40, wherein the method further comprises a second bacterial strain.

Embodiment 42. A bacterial strain produced by the method of any one of embodiments 1-40.

Embodiment 43. A composition comprising the bacterial strain of embodiment 42.

Embodiment 44. A bacterial strain comprising

• • (i) a first nucleic acid sequence encoding a first activator polypeptide that is capable of activating a quorum sensing system, wherein the first nucleic acid sequence is operably linked to an activatable promoter;
  • (ii) a second nucleic acid sequence encoding a second activator polypeptide that is capable of activating a quorum sensing system, wherein the second nucleic acid sequence is operably linked to an activatable promoter; and
  • (iii) a third nucleic acid sequence encoding a third activator polypeptide that is capable of activating a quorum sensing system, wherein the third nucleic acid sequence is operably linked to a promoter.

Embodiment 45. The bacterial strain of embodiment 44, wherein the quorum sensing system is a Lux-like quorum sensing system selected from the group consisting of: a lux system from Vibrio fischeri; a las system from Pseduomonas aeruginosa; a rhl system from Pseduomonas aeruginosa; a tra system from Agrobacterium tumefaciens; a rpa system from Rhodopseudomonas palustris; an ahy system from Aeromonas hydrophilia; a sma system from Serratia marcescens; cer system from Rhodobacter sphaeroides; and an exp system from Sinorhizobium meliloti.

Embodiment 46. The bacterial strain of embodiment 45, wherein the quorum sensing system is an rpa quorum sensing system.

Embodiment 47. The bacterial strain of embodiment 45, wherein the quorum sensing system is a lux quorum sensing system.

Embodiment 48. The bacterial strain of any one of embodiments 44-47, wherein the quorum sensing system combines elements of the rpa quorum sensing system and the lux quorum sensing system.

Embodiment 49. The bacterial strain of any one of embodiments 44-48, wherein the activatable promoter comprises a LuxR-AHL activatable luxI promoter.

Embodiment 50. The bacterial strain of any one of embodiments 44-48, wherein the activatable promoter comprises a RpaR-AHL activatable RpaI promoter.

Embodiment 51 The bacterial strain of any one of embodiments 44-50, wherein the first polypeptide comprises a first enzyme, wherein expression of the enzyme produces a quorum sensing molecule precursor.

Embodiment 52. The bacterial strain of embodiment 51, wherein the second enzyme comprises a p-coumaric acid-CoA ligase.

Embodiment 53. The bacterial strain of embodiment 52, wherein the p-coumaric acid-CoA ligase is encoded by the 4CL2nt gene from the plant Nicotiana tabacum.

Embodiment 54. The bacterial strain of any one of embodiment 44-53, wherein the quorum sensing molecule precursor is p-Coumaroyl-CoA.

Embodiment 55. The bacterial strain of any one of embodiments 44-54, wherein the second activator polypeptide comprises a second enzyme, wherein expression of the enzyme produces a quorum sensing molecule.

Embodiment 56. The bacterial strain of embodiment 55, wherein the enzyme is a 4-coumaroyl-homoserine lactone synthase (RpaI) synthase.

Embodiment 57. The bacterial strain of embodiment 56, wherein the RpaI synthase is encoded by the RpaI gene from the plant Rhodopseudomonas palustris.

Embodiment 58. The bacterial strain of any one of embodiment 55-57, wherein the quorum sensing molecule is p-Coumaroyl-HSL.

Embodiment 59. The bacterial strain of any one of embodiments 44-58, wherein the third activator polypeptide comprises a signaling molecule, wherein the signaling molecule is capable of interacting with the quorum sensing molecule.

Embodiment 60. The bacterial strain of embodiment 59, wherein the signaling molecule is a HTH-type quorum sensing-dependent transcriptional regulator (RpaR).

Embodiment 61. The bacterial strain of embodiment 60, wherein the RpaR signaling molecule is encoded by the RpaR gene from the plant Rhodopseudomonas palustris.

Embodiment 62. The bacterial strain of any one of embodiments 44-61, wherein the bacterial strain further comprises a fourth nucleic acid sequence encoding a gene of interest, wherein the fourth nucleic acid sequence is operably linked to a promoter.

Embodiment 63. The bacterial strain of embodiment 62, wherein the gene of interest encodes a bacteriophage lytic protein capable of forming a lesion in a host cell's membrane.

Embodiment 64. The bacterial strain of embodiment 63, wherein the lysis gene is E from a bacteriophage ΦX174.

Embodiment 65. The bacterial strain of any one of embodiments 62-64, wherein the gene of interest encodes a therapeutic agent.

Embodiment 66. The bacterial strain of embodiment 65, wherein the therapeutic agent is selected from the group consisting of: an inhibitory nucleic acid, a cytokine, a fusion protein, and an antibody or antigen-binding fragment thereof.

Embodiment 67. The bacterial strain of any one of embodiments 27-28, wherein the therapeutic agent is a therapeutic polypeptide.

Embodiment 68. The bacterial strain of any one of embodiments 44-67, wherein the method further comprises a fifth nucleic acid sequence encoding a reporter polypeptide selected from a gene encoding a green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), or a variant thereof.

Embodiment 69. The bacterial strain of embodiment 68, wherein the fifth nucleic acid sequence is operably linked to a promoter or is operably linked to the first, second, third, and/or fourth nucleic acid sequences.

Embodiment 70. A pharmaceutical composition comprising any of the bacterial strains of embodiments 44-69.

Embodiment 71. The pharmaceutical composition of embodiment 70, wherein the pharmaceutical composition is formulated for in situ drug delivery.

Embodiment 72. A method of treating a disease in a subject, the method comprising:

administering to a subject in need therapeutically effective amounts of any of the bacterial strains of embodiments 44-69 or a pharmaceutical composition of any one of embodiments 70-71, to thereby treat the disease in the subject.

Embodiment 73. The method of embodiment 72, wherein the disease is cancer.

It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence Appendix
SEQ
ID
NO:	Identifier	SEQUENCES
1	4c12nt gene	ATGGAGAAAGACACGAAGCAAGTTGACATCATTTTTCGCTCGAAACTG
		CCGGACATTTACATTCCGAATCATCTGCCGCTGCATAGCTACTGCTTCG
		AGAACATTTCTGAATTTTCTAGCCGTCCGTGTCTGATTAACGGTGCCAA
		TAAACAGATCTATACGTACGCGGACGTCGAGTTGAACAGCCGTAAGGT
		CGCAGCGGGTCTGCACAAGCAAGGCATCCAGCCTAAAGATACCATCAT
		GATTCTGTTGCCAAATTCTCCGGAGTTTGTGTTTGCGTTTATCGGCGCAA
		GCTACCTGGGTGCGATTAGCACGATGGCAAATCCGCTGTTTACCCCGGC
		TGAGGTTGTTAAACAAGCAAAAGCCAGCAGCGCGAAGATCATCGTGAC
		CCAAGCATGCCACGTCAACAAAGTTAAGGACTATGCCTTCGAAAATGA
		CGTCAAGATCATTTGCATCGATAGCGCGCCTGAAGGTTGTCTGCATTTC
		AGCGTTCTGACGCAGGCTAACGAACACGATATTCCGGAAGTTGAGATTC
		AGCCGGACGATGTGGTGGCCCTGCCGTACTCCAGCGGTACCACCGGCCT
		GCCGAAAGGCGTTATGCTGACCCACAAGGGCCTGGTGACGAGCGTCGC
		CCAGCAGGTCGATGGTGAAAACCCGAACCTGTACATCCACAGCGAAGA
		TGTTATGCTGTGTGTTCTGCCACTGTTCCACATCTATTCCCTGAACAGCG
		TCCTGCTGTGCGGCCTGCGTGTGGGCGCTGCCATTTTGATTATGCAGAA
		GTTTGACATTGTCAGCTTCTTGGAACTGATCCAACGCTACAAGGTGACG
		ATCGGTCCGTTCGTCCCGCCGATTGTTTTGGCCATTGCAAAAAGCCCAA
		TGGTGGATGACTATGACCTGTCGAGCGTGCGTACCGTGATGTCCGGTGC
		AGCGCCGCTGGGCAAAGAGCTGGAGGATACCGTTCGTGCGAAGTTTCC
		GAATGCGAAACTGGGTCAAGGCTACGGTATGACTGAAGCAGGTCCGGT
		GCTGGCGATGTGCTTGGCGTTCGCGAAAGAGCCGTTCGAAATCAAAAG
		CGGTGCGTGCGGTACCGTGGTGCGTAATGCTGAAATGAAAATTGTGGAT
		CCGAAAACCGGCAACAGCCTGCCGCGCAACCAGAGCGGTGAGATTTGT
		ATTCGCGGTGACCAGATTATGAAGGGCTACCTGAATGACCCGGAGGCC
		ACTGCGCGTACGATCGACAAAGAGGGTTGGCTGTATACCGGCGACATC
		GGTTATATCGATGACGACGACGAGCTGTTCATCGTTGATCGCCTGAAAG
		AGTTGATTAAGTACAAGGGTTTCCAAGTTGCGCCTGCGGAACTGGAGGC
		TCTGCTGTTGAATCATCCGAACATTAGCGATGCAGCAGTCGTTCCGATG
		AAGGATGAGCAGGCGGGTGAAGTTCCGGTCGCGTTTGTTGTGCGTAGC
		AACGGCAGCACGATCACCGAGGATGAGGTAAAGGATTTCATTTCCAAA
		CAAGTCATCTTCTATAAGCGTATCAAGCGTGTGTTTTTCGTCGATGCAAT
		CCCGAAAAGCCCGTCCGGTAAGATCCTGCGCAAAGACTTGCGTGCGAA
		GCTGGCGGCAGGTCTGCCGAATTAG
2	Promoter	TTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAA
	J23106	GCACTAGT
3	UTR region	TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCTGGATCCAAGAAGG
		AGATATAACC
4	Terminator	GCAGCGAACGACGAAAATTACGCCCTTGCAGCGTAACAGATAAAAAAA
		ATCCTTAGCTTTCGCTAAGGATGATTTCT
5	p15A origin	AACAACTTATATCGTATGGGGCTGACTTCAGGTGCTACATTTGAAGAGA
		TAAATTGCACTGAAATCTAGAAATATTTTATCTGATTAATAAGATGATC
		TTCTTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAA
		AAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTC
		TTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTGTC
		CTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTA
		AATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGG
		GTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTG
		AACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTACCC
		GGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACAGCG
		GAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGG
		GAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTC
		GCCACCACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGCG
		GAGCCTATGGAAAAACGGCTTTGCCGCGGCCCTCTCACTTCCCTGTTAA
		GTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCATT
		TCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGAG
		GAAGCGGAATATATCC
6	Plux	TTTGATTCTAATAAATTGGATTTTTGTCACACTATTGTATCGCTGGGAAT
	Promoter	ACAATTACTTAACATAAGCACCTGTAGGATCGTACAGGTTTACGCAAGA
		AAATGGTTTGTTATAGTCGAAT
7	Lysine gene	ATGGTACGCTGGACTTTGTGGGATACCCTCGCTTTCCTGCTCCTGTTGAG
	XI74E	TTTATTGCTGCCGTCATTGCTTATTATGTTCATCCCGTCAACATTCAAAC
		GGCCTGTCTCATCATGGAAGGCGCTGAATTTACGGAAAACATTATTAAT
		GGCGTCGAGCGTCCGGTTAAAGCCGCTGAATTGTTCGCGTTTACCTTGC
		GTGTACGCGCAGGAAACACTGACGTTCTTACTGACGCAGAAGAAAACG
		TGCGTCAAAAATTACGTGCGGAAGGAGTGA
8	RpaR gene	ATGATCGTCGGCGAAGATCAGCTTTGGGGACGGCGTGCGCTGGAATTC
		GTCGATTCCGTCGAACGGCTCGAGGCGCCGGCGCTGATCAGCCGGTTCG
		AATCGCTGATCGCGAGCTGCGGATTTACCGCCTACATCATGGCCGGCCT
		GCCGTCGCGCAATGCCGGACTACCGGAGCTGACGCTGGCCAATGGCTG
		GCCGCGAGACTGGTTCGATCTGTATGTCAGCGAAAACTTCAGCGCGGTC
		GATCCGGTGCCGCGCCACGGCGCTACCACGGTTCATCCTTTCGTATGGT
		CCGATGCACCCTACGACCGCGACCGTGATCCGGCCGCCCACCGGGTCAT
		GACCCGGGCGGCGGAATTCGGACTGGTCGAGGGTTACTGCATTCCGCTG
		CACTACGACGACGGTAGCGCCGCGATCAGCATGGCCGGCAAGGATCCG
		GACCTCAGCCCGGCCGCGCGCGGCGCGATGCAGCTGGTCAGCATCTAC
		GCGCATAGTCGCCTGCGCGCACTCAGCCGGCCAAAGCCGATCCGGCGC
		AACCGGCTCACGCCGCGCGAGTGCGAGATCCTGCAATGGGCAGCGCAG
		GGCAAGACCGCCTGGGAAATCTCGGTAATCCTCTGCATCACCGAACGC
		ACGGTGAAATTCCATCTGATCGAAGCCGCCCGCAAGCTCGACGCCGCC
		AACCGCACCGCGGCGGTTGCCAAGGCATTGACGCTCGGATTGATCCGTT
		TGTGA
9	Chloramphenicol	GATATCTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAAC
	resistance	TTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTAT
	gene	CGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTG
		GATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGA
		GGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTG
		GATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTT
		ATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAA
		TTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTC
		ACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTC
		TGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGC
		AAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTT
		TATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCA
		gttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttc
		ACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTG
		GCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAA
		TGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGT
		AATTTGATATC
10	>pTD103LuxI_CFP	CTCGAGTTAATTTTTAAAGTATGGGCAATCAATTGCTCCTGTTAAAATT
		GCTTTAGAAATACTTTGGCAGCGGTTTGTTGTATTGAGTTTCATTTGCGC
		ATTGGTTAAATGGAAAGTGACAGTACGCTCACTGCAGCCTAATATTTTT
		GAAATATCCCAAGAGCTTTTTCCTTCGCATGCCCACGCTAAACATTCTTT
		TTCTCTTTTGGTTAAATCGTTGTTTGATTTATTATTTGCTATATTTATTTT
		TCGATAATTATCAACTAGAGAAGGAACAATTAATGGTATGTTCATACAC
		GCATGTAAAAATAAACTATCTATATAGTTGTCTTTTTCTGAATGTGCAA
		AACTAAGCATTCCGAAGCCATTGTTAGCCGTATGAATAGGGAAACTAA
		ACCCAGTGATAAGACCTGATGTTTTCGCTTCTTTAATTACATTTGGAGAT
		TTTTTATTTACAGCATTGTTTTCAAATATATTCCAATTAATTGGTGAATG
		ATTGGAGTTAGAATAATCTACTATAGGATCATATTTTATTAAATTAGCG
		TCATCATAATATTGCCTCCATTTTTTAGGGTAATTATCTAGAATTGAAAT
		ATCAGATTTAACCATAGAATGAGGATAAATGATCGCGAGTAAATAATA
		TTCACAATGTACCATTTTAGTCATATCAGATAAGCATTGATTAATATCAT
		TATTGCTTCTACAAGCTTTAATTTTATTAATTATTCTGTATGTGTCGTCG
		GCATTTATGTTTTTCATACCCATCTCTTTATCCTTACCTATTGTTTGTCGC
		AAGTTTTGCGTGTTATATATCATTAAAACGGTAATGGATTGACATTTGA
		TTCTAATAAATTGGATTTTTGTCACACTATTGTATCGCTGGGAATACAAT
		TACTTAACATAAGCACCTGTAGGATCGTACAGGTTTACGCAAGAAAATG
		GTTTGTTATAGTCGAATGAATTCATTAAAGAGGAGAAAGGTACCATGAC
		TATAATGATAAAAAAATCGGATTTTTTGGCAATTCCATCGGAGGAGTAT
		AAAGGTATTCTAAGTCTTCGTTATCAAGTGTTTAAGCAAAGACTTGAGT
		GGGACTTAGTTGTAGAAAATAACCTTGAATCAGATGAGTATGATAACTC
		AAATGCAGAATATATTTATGCTTGTGATGATACTGAAAATGTAAGTGGA
		TGCTGGCGTTTATTACCTACAACAGGTGATTATATGCTGAAAAGTGTTT
		TTCCTGAATTGCTTGGTCAACAGAGTGCTCCCAAAGATCCTAATATAGT
		CGAATTAAGTCGTTTTGCTGTAGGTAAAAATAGCTCAAAGATAAATAAC
		TCTGCTAGTGAAATTACAATGAAACTATTTGAAGCTATATATAAACACG
		CTGTTAGTCAAGGTATTACAGAATATGTAACAGTAACATCAACAGCAAT
		AGAGCGATTTTTAAAGCGTATTAAAGTTCCTTGTCATCGTATTGGAGAC
		AAAGAAATTCATGTATTAGGTGATACTAAATCGGTTGTATTGTCTATGC
		CTATTAATGAACAGTTTAAAAAAGCAGTCTTAAATGCAGCGAACGACG
		AAAATTACGCCCTTGCAGCGTAAACGCGTGCTAGAGGCATCAAATAAA
		ACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTG
		TCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGCCCTAGACCTAGG
		CGTCTTCACCTCGAGTTAATTTTTAAAGTATGGGCAATCAATTGCTCCTG
		TTAAAATTGCTTTAGAAATACTTTGGCAGCGGTTTGTTGTATTGAGTTTC
		ATTTGCGCATTGGTTAAATGGAAAGTGACAGTACGCTCACTGCAGCCTA
		ATATTTTTGAAATATCCCAAGAGCTTTTTCCTTCGCATGCCCACGCTAAA
		CATTCTTTTTCTCTTTTGGTTAAATCGTTGTTTGATTTATTATTTGCTATA
		TTTATTTTTCGATAATTATCAACTAGAGAAGGAACAATTAATGGTATGT
		TCATACACGCATGTAAAAATAAACTATCTATATAGTTGTCTTTTTCTGAA
		TGTGCAAAACTAAGCATTCCGAAGCCATTGTTAGCCGTATGAATAGGGA
		AACTAAACCCAGTGATAAGACCTGATGTTTTCGCTTCTTTAATTACATTT
		GGAGATTTTTTATTTACAGCATTGTTTTCAAATATATTCCAATTAATTGG
		TGAATGATTGGAGTTAGAATAATCTACTATAGGATCATATTTTATTAAA
		TTAGCGTCATCATAATATTGCCTCCATTTTTTAGGGTAATTATCTAGAAT
		TGAAATATCAGATTTAACCATAGAATGAGGATAAATGATCGCGAGTAA
		ATAATATTCACAATGTACCATTTTAGTCATATCAGATAAGCATTGATTA
		ATATCATTATTGCTTCTACAAGCTTTAATTTTATTAATTATTCTGTATGT
		GTCGTCGGCATTTATGTTTTTCATACCCATCTCTTTATCCTTACCTATTGT
		TTGTCGCAAGTTTTGCGTGTTATATATCATTAAAACGGTAATGGATTGA
		CATTTGATTCTAATAAATTGGATTTTTGTCACACTATTGTATCGCTGGGA
		ATACAATTACTTAACATAAGCACCTGTAGGATCGTACAGGTTTACGCAA
		GAAAATGGTTTGTTATAGTCGAATGAATTCATTAAAGAGGAGAAAGGT
		ACCATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGTCCCAATTTTGG
		TTGAATTAGATGGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGA
		AGGTGAAGGTGATGCTACTTACGGTAAATTGACCTTAAAATTTATTTGT
		ACTACTGGTAAATTGCCAGTTCCATGGCCAACCTTAGTCACTACTTTAA
		CTTGGGGTGTTCAATGTTTTGCTAGATACCCAGATCATATGAAACAACA
		TGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAGAAAGAACT
		ATTTTTTTCAAAGATGACGGTAACTACAAGACCAGAGCTGAAGTCAAGT
		TTGAAGGTGATACCTTAGTTAATAGAATCGAATTAAAAGGTATTGATTT
		TAAAGAAGATGGTAACATTTTAGGTCACAAATTGGAATACAACGCTATT
		TCTGATAATGTTTACATCACTGCTGACAAACAAAAGAATGGTATCAAAG
		CTAACTTCAAAATTAGACACAACATTGAAGATGGTGGTGTTCAATTAGC
		TGACCATTATCAACAAAATACTCCAATTGGTGATGGTCCAGTCTTGTTA
		CCAGACAACCATTACTTATCCACTCAATCTAAATTATCCAAAGATCCAA
		ACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTACTGCTGCTGG
		TATTACCCATGGTATGGATGAATTGTACAAAACTAGTGCAGCGAACGAC
		GAAAATTACGCCCTTGCAGCGTGAAAGCTTAATTAGCTGATCTAGACGC
		GTGCTAGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGC
		CTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAA
		ATCCGCCGCCCTAGACCTAGGGCGTTCGGCTGCGGCGAGCGGTATCAGC
		TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC
		AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTA
		AAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA
		GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG
		ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT
		CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTC
		GGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG
		GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC
		AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC
		GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGAT
		TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG
		GCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTG
		CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA
		AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT
		TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACG
		GGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCA
		TGACTAGTGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCG
		AGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTA
		TCAACAGGAGTCCAAGCGAGCTCTCGAACCCCAGAGTCCCGCTCAGAA
		GAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGC
		GGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAG
		CTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCC
		GCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTT
		TCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGA
		TCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTG
		GCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACC
		GGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGG
		TCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCA
		TCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGG
		AGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTT
		CAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCA
		GCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGA
		CAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCG
		GAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAG
		CCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCAT
		CTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGATCTT
		GATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTA
		CTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTC
		CGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGG
		CGTATCACGAGGCCCTTTCGTCTTCAC
11	>(3)	TCCTCATCCTGTCTCTTGATCAGATCTTGATCCCCTGCGCCATCAGATCC
	pTD103-	TTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACCTT
	RpaR-RpaI-	ACCAGAGGGCGCCCCAGCTGGCAATTCCGACGTCTAAGAAACCATTATT
	LAA-sfGFP	ATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
		TTCACCTCGAGTCACAAACGGATCAATCCGAGCGTCAATGCCTTGGCAA
		CCGCCGCGGTGCGGTTGGCGGCGTCGAGCTTGCGGGCGGCTTCGATCAG
		ATGGAATTTCACCGTGCGTTCGGTGATGCAGAGGATTACCGAGATTTCC
		CAGGCGGTCTTGCCCTGCGCTGCCCATTGCAGGATCTCGCACTCGCGCG
		GCGTGAGCCGGTTGCGCCGGATCGGCTTTGGCCGGCTGAGTGCGCGCA
		GGCGACTATGCGCGTAGATGCTGACCAGCTGCATCGCGCCGCGCGCGG
		CCGGGCTGAGGTCCGGATCCTTGCCGGCCATGCTGATCGCGGCGCTACC
		GTCGTCGTAGTGCAGCGGAATGCAGTAACCCTCGACCAGTCCGAATTCC
		GCCGCCCGGGTCATGACCCGGTGGGCGGCCGGATCACGGTCGCGGTCG
		TAGGGTGCATCGGACCATACGAAAGGATGAACCGTGGTAGCGCCGTGG
		CGCGGCACCGGATCGACCGCGCTGAAGTTTTCGCTGACATACAGATCGA
		ACCAGTCTCGCGGCCAGCCATTGGCCAGCGTCAGCTCCGGTAGTCCGGC
		ATTGCGCGACGGCAGGCCGGCCATGATGTAGGCGGTAAATCCGCAGCT
		CGCGATCAGCGATTCGAACCGGCTGATCAGCGCCGGCGCCTCGAGCCG
		TTCGACGGAATCGACGAATTCCAGCGCACGCCGTCCCCAAAGCTGATCT
		TCGCCGACGATCATACTATTGTATCGCTGGGAATACAATTACTTAACAT
		AAGCACCTGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATA
		GTCGAATGAATTCATTAAAGAGGAGAAAGGTACCATGCGTAAAGGCGA
		AGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGAT
		GTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCA
		ACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGC
		CGGTACCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTG
		CTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCC
		GCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATG
		ACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCC
		TGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCA
		ATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTA
		CATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAAT
		TCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCA
		GCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCAC
		TATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGC
		GATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATG
		GTATGGATGAACTGTACAAATGAAAGCTTAATTAGCTGATCTAGACGCG
		TGCTAGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCC
		TTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAA
		TCCGCCGCCCTAGACCTAGCCGTCTTCACCATGCATTCACAAACGGATC
		AATCCGAGCGTCAATGCCTTGGCAACCGCCGCGGTGCGGTTGGCGGCGT
		CGAGCTTGCGGGCGGCTTCGATCAGATGGAATTTCACCGTGCGTTCGGT
		GATGCAGAGGATTACCGAGATTTCCCAGGCGGTCTTGCCCTGCGCTGCC
		CATTGCAGGATCTCGCACTCGCGCGGCGTGAGCCGGTTGCGCCGGATCG
		GCTTTGGCCGGCTGAGTGCGCGCAGGCGACTATGCGCGTAGATGCTGAC
		CAGCTGCATCGCGCCGCGCGCGGCCGGGCTGAGGTCCGGATCCTTGCCG
		GCCATGCTGATCGCGGCGCTACCGTCGTCGTAGTGCAGCGGAATGCAGT
		AACCCTCGACCAGTCCGAATTCCGCCGCCCGGGTCATGACCCGGTGGGC
		GGCCGGATCACGGTCGCGGTCGTAGGGTGCATCGGACCATACGAAAGG
		ATGAACCGTGGTAGCGCCGTGGCGCGGCACCGGATCGACCGCGCTGAA
		GTTTTCGCTGACATACAGATCGAACCAGTCTCGCGGCCAGCCATTGGCC
		AGCGTCAGCTCCGGTAGTCCGGCATTGCGCGACGGCAGGCCGGCCATG
		ATGTAGGCGGTAAATCCGCAGCTCGCGATCAGCGATTCGAACCGGCTG
		ATCAGCGCCGGCGCCTCGAGCCGTTCGACGGAATCGACGAATTCCAGC
		GCACGCCGTCCCCAAAGCTGATCTTCGCCGACGATCATACTATTGTATC
		GCTGGGAATACAATTACTTAACATAAGCACCTGTAGGATCGTACAGGTT
		TACGCAAGAAAATGGTTTGTTATAGTCGAATGAATTCATTAAAGAGGA
		GAAAGGTACCATGCAGGTTCATGTCATCCGTCGAGAGAACCGCGCGCT
		CTATGCCGGTCTGCTCGAAAAGTACTTCCGCATCCGTCACCAGATCTAC
		GTCGTCGAGCGCGGCTGGAAGGAGCTCGATCGGCCGGATGGCCGCGAG
		ATCGATCAGTTCGACACCGAAGACGCCGTGTATCTGCTCGGCGTCGACA
		ATGACGACATCGTCGCCGGCATGCGGATGGTGCCGACCACGTCACCGA
		CGCTCCTCAGCGACGTCTTCCCGCAGCTTGCGCTGGCAGGCCCGGTGCG
		GCGGCCGGATGCCTACGAGCTGTCGCGGATCTTCGTGGTACCGCGCAAG
		CGCGGCGAGCATGGCGGCCCGCGCGCCGAAGCCGTGATCCAGGCGGCC
		GCGATGGAGTACGGCCTGTCGATCGGTCTGTCGGCCTTCACCATCGTGC
		TGGAGACCTGGTGGCTGCCGCGACTGGTGGACCAGGGCTGGAAGGCAA
		AGCCGCTCGGCCTGCCTCAGGACATCAACGGATTCTCGACCACCGCAGT
		GATCGTCGACGTCGACGACGACGCCTGGGTCGGCATCTGCAATCGCCGC
		TCGGTGCCCGGACCCACGCTGGAATGGCGCGGGCTCGAAGCCATCCGC
		CGTCATTCGCTTCCGGAATTCCAGGTGATTTCAGCAGCGAACGACGAAA
		ATTACGCCCTTGCAGCGTAAGTGCTAGAGGCATCAAATAAAACGAAAG
		GCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAA
		CGCTCTCCTGAGTAGGACAAATCCGCCGCCCTAGACCTAGGGCGTTCGG
		CTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCA
		CAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG
		CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT
		AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG
		AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT
		GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT
		ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCA
		CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT
		GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA
		CTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCA
		GCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCT
		ACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACA
		GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAG
		TTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTT
		TTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGA
		AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAAC
		TCACGTTAAGGGATTTTGGTCATGACTAGTGCTTGGATTCTCACCAATA
		AAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGT
		TCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCTCGA
		ACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGC
		GATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCG
		GTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAAC
		GCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGA
		ATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATC
		GCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGC
		CTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGAT
		CATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGAT
		GCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTA
		TGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAG
		CAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCA
		GCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGG
		AACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGT
		TCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGC
		CCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTC
		TGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAG
		AACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGA
12	>pZA35\X174\	TTAATTTTTAAAGTATGGGCAATCAATTGCTCCTGTTAAAATTGCTTTAG
	(+Lux	AAATACTTTGGCAGCGGTTTGTTGTATTGAGTTTCATTTGCGCATTGGTT
		AAATGGAAAGTGACAGTACGCTCACTGCAGCCTAATATTTTTGAAATAT
		CCCAAGAGCTTTTTCCTTCGCATGCCCACGCTAAACATTCTTTTTCTCTT
		TTGGTTAAATCGTTGTTTGATTTATTATTTGCTATATTTATTTTTCGATAA
		TTATCAACTAGAGAAGGAACAATTAATGGTATGTTCATACACGCATGTA
		AAAATAAACTATCTATATAGTTGTCTTTTTCTGAATGTGCAAAACTAAG
		CATTCCGAAGCCATTGTTAGCCGTATGAATAGGGAAACTAAACCCAGTG
		ATAAGACCTGATGTTTTCGCTTCTTTAATTACATTTGGAGATTTTTTATT
		TACAGCATTGTTTTCAAATATATTCCAATTAATTGGTGAATGATTGGAG
		TTAGAATAATCTACTATAGGATCATATTTTATTAAATTAGCGTCATCATA
		ATATTGCCTCCATTTTTTAGGGTAATTATCTAGAATTGAAATATCAGATT
		TAACCATAGAATGAGGATAAATGATCGCGAGTAAATAATATTCACAAT
		GTACCATTTTAGTCATATCAGATAAGCATTGATTAATATCATTATTGCTT
		CTACAAGCTTTAATTTTATTAATTATTCTGTATGTGTCGTCGGCATTTAT
		GtttttCATACCCATCTCTTTATCCTTACCTATTGTTTGTCGCAAGTTTTG
		CGTGTTATATATCATTAAAACGGTAATGGATTGACATTTGATTCTAATA
		AATTGGATTTTTGTCACACTATTGTATCGCTGGGAATACAATTACTTAAC
		ATAAGCACCTGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTA
		TAGTCGAATAAACGCAAGGGAGGTTGGTATGGTACGCTGGACTTTGTG
		GGATACCCTCGCTTTCCTGCTCCTGTTGAGTTTATTGCTGCCGTCATTGC
		TTATTATGTTCATCCCGTCAACATTCAAACGGCCTGTCTCATCATGGAA
		GGCGCTGAATTTACGGAAAACATTATTAATGGCGTCGAGCGTCCGGTTA
		AAGCCGCTGAATTGTTCGCGTTTACCTTGCGTGTACGCGCAGGAAACAC
		TGACGTTCTTACTGACGCAGAAGAAAACGTGCGTCAAAAATTACGTGC
		GGAAGGAGTGAACGCGTGCTAGAGGCATCAAATAAAACGAAAGGCTCA
		GTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTC
		TCCTGAGTAGGACAAATCCGCCGCCCTAGACCTAGGGGATATATTCCGC
		TTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCG
		GAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAG
		ATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATA
		GGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTG
		GTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG
		CGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCA
		TTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTC
		CGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTT
		CAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACC
		CGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGAT
		TTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAA
		GGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAA
		AGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGT
		TTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAA
		GAAGATCATCTTATTAATCAGATAAAATATTTCTAGATTTCAGTGCAAT
		TTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTT
		GTTACTAGTGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCG
		AGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTA
		TCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCC
		ACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAA
		GCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCAC
		CTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAG
		AAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCC
		AGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGA
		AATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTG
		TAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAA
		CGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCC
		CATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAG
		CATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGT
		GCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACG
		GTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTT
		CTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTT
		TTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAA
		TACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTT
		ACGTGCCGATCAACGTCTCATTTTCGCCAGATATCGACGTC

Claims

1. A method of inducing quorum sensing, the method comprising: (a) culturing a bacterial strain, wherein the bacterial strain comprises (i) a first nucleic acid sequence encoding a first activator polypeptide, wherein expression of the first activator polypeptide results in production of a quorum sensing molecule precursor, wherein the first nucleic acid sequence is operably linked to a promoter;(ii) a second nucleic acid sequence encoding a second activator polypeptide, wherein expression of the second activator polypeptide results in production of a quorum sensing molecule, wherein the second nucleic acid sequence is operably linked to a first activatable promoter;(iii) a third nucleic acid sequence encoding a third activator polypeptide, wherein expression of the third activator polypeptide results in activation of the quorum sensing system, wherein the third nucleic acid sequence is operably linked to a second activatable promoter; and(iv) a fourth nucleic acid sequence encoding a gene of interest, wherein the fourth nucleic acid sequence is operably linked to a third activatable promoter; and(b) contacting the bacterial strain with an inducer molecule; and(c) converting the inducer molecule into a quorum sensing molecule, thereby allowing induction of quorum sensing.

2. The method of claim 1, wherein the quorum sensing system comprises a Lux-like quorum sensing system selected from the group consisting of: a rpa system from Rhodopseudomonas palustris; a lux system from Vibrio fischeri; a las system from Pseduomonas aeruginosa; a rhl system from Pseduomonas aeruginosa; a tra system from Agrobacterium tumefaciens; a rpa system from Rhodopseudomonas palustris; an ahy system from Aeromonas hydrophilia; a sma system from Serratia marcescens; cer system from Rhodobacter sphaeroides; and an exp system from Sinorhizobium meliloti.

3. The method of claim 2, wherein the quorum sensing system comprises all or part of an rpa quorum sensing system, wherein the rpa sensing system is from Rhodopseudomonas palustris.

4. The method of claim 1, wherein the second activator polypeptide comprises a luxI homolog selected from the group consisting of: RpaI, RhlI, BjaI, AubI, CerI, LasI, BraI, SinI, LuxI, and EsaI; and wherein the third activator polypeptide comprises a luxR homolog selected from the group consisting of: RpaR, AubR, BjaR, LasR, RhlR, LuxR and TraR.

5. The method of claim 1, wherein (i) the first activatable promoter operably linked to the second nucleic acid is a promoter comprising one or more lux box-like sequences;(ii) the second activatable promoter operably linked to the third nuleic acid is a promoter comprising one or more lux box-like sequences; and/or(iii) the third activatable promoter operably linked to the fourth nucleic acid is a promoter comprising one or more lux box-like sequences.

6. The method of claim 1, wherein the first activator polypeptide comprises a p-coumaric acid-CoA ligase, wherein the p-coumaric acid-CoA ligase is encoded by the 4CL2nt gene from the plant Nicotiana tabacum.

7. The method of claim 1, wherein the quorum sensing molecule precursor is p-Coumaroyl-CoA.

8. The method of claim 1, wherein the second activator polypeptide is a 4-coumaroyl-homoserine lactone synthase (RpaI) synthase, wherein the RpaI synthase is encoded by the RpaI gene from the plant Rhodopseudomonas palustris.

9. The method of claim 1, wherein the quorum sensing molecule is p-Coumaroyl-HSL.

10. The method of claim 1, wherein the third activator polypeptide comprises a signaling molecule, wherein the signaling molecule interacts with the quorum sensing molecule.

11. The method of claim 1, wherein the signaling molecule is a HTH-type quorum sensing-dependent transcriptional regulator (RpaR), wherein the RpaR signaling molecule is encoded by the RpaR gene from the plant Rhodopseudomonas palustris.

12. The method of claim 1, wherein the interaction of RpaR and the quorum sensing molecule p-Coumaroyl-HSL results in transcriptional regulation of the fourth nucleic acid sequence.

13. The method of claim 1, wherein the inducer molecule is p-coumaric acid.

14. The method of claim 1, wherein the gene of interest encodes a bacteriophage lytic protein capable of forming a lesion in a host cell's membrane.

15. The method of claim 1, wherein the method further comprises one or more additional bacterial strains.

16. A bacterial strain produced by the method of claim 1.

17. A set of nucleic acids comprising the nucleic acid sequences of claim 1.

18. A composition comprising the bacterial strain of claim 16, the one or more bacterial strains of claim 15, or the set of nucleic acid sequences of claim 17.

19. A method of treating a disease in a subject, the method comprising: administering to a subject in need thereof a therapeutically effective amount of the bacterial strain of claim 16 or the composition of claim 18, to thereby treat the disease in the subject.

20. The method of claim 19, wherein the disease is cancer.