AUXOTROPHIC SELECTION SYSTEM

Abstract

A method for the analysis of microorganisms, which produce a compound, the method comprising: a. providing a microorganism which produces a compound of interest and a detector microorganism which comprises a reporter gene or reporter gene operon, wherein the microorganism producing said compound of interest and the detector microorganism are combined into single droplets, wherein each droplet comprises at least one cell of each strain; b. subjecting the droplets to a microfluidic system; c. analyzing the droplets for the activation of the reporter gene of the detector strain; d. sorting and collecting the droplets comprising the detector microorganism with expressed reporter gene.

Claims

1. A method for the analysis of microorganisms, which produce a compound of interest, the method comprising: a. providing a microorganism which produces a compound of interest and a detector microorganism which comprises a reporter gene or reporter gene operon, wherein the microorganism producing said compound of interest and the detector microorganism are combined into single droplets, wherein each droplet comprises at least one cell of each strain; b. subjecting the droplets to a microfluidic system; c. analyzing the droplets for the activity of the reporter gene of the detector strain; d. sorting and collecting the droplets comprising the detector microorganism with expressed reporter gene.

2. The method according to claim 1, wherein the microorganism producing a compound and/or the detector microorganism is a bacterial, fungal, yeast, algal, eukaryotic, prokaryotic or insect strain.

3. The method according to claims 1 or 2, wherein the reporter gene product produces a fluorescent signal.

4. The method according to any of the claims 1 to 3, wherein the reporter gene encodes a fluorescent protein such as green fluorescent protein (GFP), a variant of GFP, yellow fluorescent protein (YFP), a variant of YFP, red fluorescent protein (RFP), a variant of RFP, cyan fluorescent protein (CFP), a variant of CFP or the reporter gene operon is a luminescence operon such as the lux operon.

5. The method according to any of the claims 1 to 4, wherein the incubation is performed in the microfluidic system.

6. The method according to claims 1 to 5, wherein the compound is a primary metabolite, including but not limited to: L- and D-amino acids; sugars and carbon sources such as L-arabinose, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, N-acetylneuraminate, lactose, D-glucosamine, D-glucose-6-phosphate, D-xylose, D-galactose, glycerol, maltose, maltotriose, and melibiose; nucleosides such as cytidine, guanine, adenine, thymidin, guanosine, adenosine; lipids such as hexadecanoate and glycerol 3-phosphate; indole, maltohexose, maltopentose, putrescine, spermidine, ornithine, tetradecanoate, and nicotinamide adenine dinucleotide or a secondary metabolite.

7. A microfluidic device capable of co-encapsulating at least two types of cells, the device comprising: a. at least one inlet for a culture medium comprising a first microorganism; b. at least one inlet for a culture medium comprising a second microorganism; c. at least one inlet for a phase immiscible with the culture media; d. a chamber for combining the first and second medium, suitable to generate droplets comprising at least one cell of each microorganism, and to encapsulate the droplets in the immiscible phase; e. optionally, means to incubate the droplets at a constant or variable temperature; f. optionally, a detector to detect the activity of a reporter gene; g. optionally, an outlet coupled with means for sorting droplets.

8. The microfluidic device according to claim 7 capable of co-encapsulating at least two types of cells, the device comprising: a. a chamber for generating droplets of the first medium, and to encapsulate the droplets in the immiscible phase; b. a chamber for generating droplets of the second medium, and to encapsulate the droplets in the immiscible phase; c. a chamber for combining droplets of the first medium with droplets of the second medium and subsequently fusing said droplets to yield larger droplets comprising a mixture of the first medium and the second medium.

9. The microfluidic device according to claim 7 capable of co-encapsulating at least two types of cells, the device comprising: a. a chamber for generating droplets of the first medium, and to encapsulate the droplets in the immiscible phase; b. a chamber for combining droplets of the first medium with the second medium by picoinjection to yield droplets comprising a mixture of the first medium with the second medium;

10. The microfluidic device according to any of claims 7 to 9, wherein the detector is a fluorescence detector.

11. The microfluidic device according to claim 10, wherein detector is a fluorescence detector and able to quantify the fluorescence intensity.

12. The microfluidic device according to any of the claims 7 to 11, wherein the detector is coupled to a computing device.

13. The microfluidic device according to any of the claims 7 to 12, wherein the device comprises means to incubate the droplets at a temperature range between 18 C. and 50 C.

14. The microfluidic device according to any of claims 7 to 13, wherein the means for sorting droplets comprise dielectric sorting of droplets.

15. Use of a microfluidic device according to any of the claims 7 to 14 in a method according to claims 1 to 6.

Description

FIGURE LEGENDS

[0177] FIGS. 1 to 3: schematic examples of preferred workflows of the method.

[0178] FIGS. 4 to 6: schematics of microfluidic devices for droplet generation. Broken lines represent positions, where the droplets might be further processed either within or of the microfluidic device.

EXAMPLES

Example 1

[0179] A strain of Escherichia coli (e.g., MG1655) is transformed with a plasmid (named here as pTrp) containing the trpABCDE operon under the control of a strong constitutive promoter. The E. coli strain harboring pTrp is able to overproduce L-tryptophan and secrete the amino acid in to the surrounding culture medium, hereafter referred to as the producer strain.

[0180] A strain of Saccharomyces cerevisiae that is auxotrophic for L-tryptophan and L-leucine (e.g., W303 and its derivatives) is transformed with a plasmid (named here as pFluor) containing the coding sequence of a fluorescent protein (e.g., GFP, eGFP, mCherry, RFP, etc.) under the control of a strong constitutive promoter (e.g., P.sub.TEF1) as well as the gene or gene operon that allows for intracellular production of L-leucine. Such complementation of the L-leucine auxotroph allows for positive selection of S. cerevisiae cells harboring the pFluor plasmid. When cultured in the presence of L-tryptophan but in the absence of L-leucine, the auxotrophic Saccharomyces cerevisiae strain harboring pFluor proliferates and expresses the fluorescent protein intracellularly. The proliferation of this strain can be monitored via fluorescence measurements, namely illuminating the cells with light of a wavelength or range of wavelengths and measuring the amount of light emitted by the cells at a wavelength or range of wavelengths greater than the wavelength(s) used for illumination. This auxotrophic Saccharomyces cerevisiae strain will be referred to hereafter as the detector strain.

[0181] The producer strain is inoculated into a minimal medium (e.g., M9 minimal medium with 4 g/L glucose). This culture is grown for 4-8 hours at 37 C. with shaking at 200 rpm, then diluted to an OD.sub.600 of 0.02 using the same minimal medium. The detector strain is inoculated into a synthetically defined medium containing L-tryptophan (to allow for cell growth) but missing L-leucine (to ensure maintenance of the pFluor plasmid). This detector strain culture is grown for 4-8 hours at 30 C. with shaking at 200 rpm. The detector strain culture is then washed with an isotonic buffer and resuspended using a synthetically defined medium missing both L-tryptophan and L-leucine. Microfluidic droplets 20 pL in volume are generated using a microfluidic system in which the aqueous phase comprising the producer strain diluted in minimal medium is separated into droplets by a fluorinated oil (e.g., HFE7500) containing a fluorinated surfactant. These microfluidic droplets are collected and subjected to picoinjection, in which a small, defined volume (5 pL) of detector strain culture is added to each microfluidic droplet, thereby contacting cells of the producer strain with cells of the detector strain within microfluidic droplets. The picoinjected droplets are then collected and incubated at 30 C. to allow for growth of the producer strain, production of L-tryptophan, subsequent growth of the detector strain, and concomitant production of the fluorescent protein.

[0182] The microfluidic droplets are then analyzed using the microfluidic system. The fluorescence of each droplet is analyzed by illuminating the droplet with a laser having a wavelength corresponding to the excitation maximum of the fluorescent protein of interest and measuring the amount of light emitted by the droplet at a range of wavelengths longer than the wavelength used for illumination/excitation. Droplets exhibiting higher fluorescence must contain higher concentrations of fluorescent protein and must therefore contain a higher number of cells of the detector strain. One may also infer that droplets containing higher numbers of detector strain cells must also contain producer strain cells which generated higher amounts of L-tryptophan.

[0183] Using the microfluidic system, droplets exhibiting high levels of fluorescence are separated from the remainder of the droplet pool and collected for further analysis.

Example 2

[0184] A strain of E. coli is engineered to overproduce L-tryptophan via replacement of the native trpABCDE promoter with a strong constitutive promoter. However, feedback regulation has been shown to limit the amount of L-tryptophan that can be produced by this engineered E. coli strain. To overcome this feedback regulation and other regulatory phenomena that may limit L-tryptophan production, the engineered strain is subjected to UV-induced random mutagenesis, generating a library of L-tryptophan-producing E. coli strains. Following generation, this library is cultured on solid medium. Prior to plating on a solid medium, the library is sufficiently diluted such that clonal isolates are obtained on solid media following a period of incubation.

[0185] A strain of Saccharomyces cerevisiae that is auxotrophic for L-tryptophan and L-leucine (e.g., W303 and its derivatives) is transformed with a plasmid (named here as pFluor) containing the coding sequence of a fluorescent protein (e.g., GFP, eGFP, mCherry, RFP, etc.) under the control of a strong constitutive promoter (e.g., P.sub.TEF1) as well as the gene or gene operon that allows for intracellular production of L-leucine. Such complementation of the L-leucine auxotroph allows for positive selection of S. cerevisiae cells harboring the pFluor plasmid. When cultured in the presence of L-tryptophan but in the absence of L-leucine, the auxotrophic Saccharomyces cerevisiae strain harboring pFluor proliferates and expresses the fluorescent protein intracellularly. The proliferation of this strain can be monitored via fluorescence measurements, namely illuminating the cells with light of a wavelength or range of wavelengths and measuring the amount of light emitted by the cells at a wavelength or range of wavelengths greater than the wavelength(s) used for illumination. This auxotrophic Saccharomyces cerevisiae strain will be referred to hereafter as the detector strain.

[0186] The producer strain library is recovered from solid medium, then diluted and inoculated into a minimal medium (e.g., M9 minimal medium with 4 g/L glucose). This culture is grown for 4-8 hours at 37 C. with shaking at 200 rpm, then diluted to an OD.sub.600 of 0.02 using the same minimal medium. The detector strain is inoculated into a synthetically defined medium containing L-tryptophan (to allow for cell growth) but missing L-leucine (to ensure maintenance of the pFluor plasmid). This detector strain culture is grown for 4-8 hours at 30 C. with shaking at 200 rpm. The detector strain culture is then washed with an isotonic buffer and resuspended using a synthetically defined medium missing both L-tryptophan and L-leucine. Microfluidic droplets 20 pL in volume are generated using a microfluidic system in which the aqueous phase comprising the producer strain diluted in minimal medium is separated into droplets by a fluorinated oil (e.g., HFE7500) containing a fluorinated surfactant. These microfluidic droplets are collected and subjected to picoinjection, in which a small, defined volume (5 pL) of detector strain culture is added to each microfluidic droplet, thereby contacting cells of the producer strain with cells of the detector strain within microfluidic droplets. The picoinjected droplets are then collected and incubated at 30 C. to allow for growth of the producer strain, production of L-tryptophan, subsequent growth of the detector strain, and concomitant production of the fluorescent protein.

[0187] The microfluidic droplets are then analyzed using the microfluidic system. The fluorescence of each droplet is analyzed by illuminating the droplet with a laser having a wavelength corresponding to the excitation maximum of the fluorescent protein of interest and measuring the amount of light emitted by the droplet at a range of wavelengths longer than the wavelength used for illumination/excitation. Droplets exhibiting higher fluorescence must contain higher concentrations of fluorescent protein and must therefore contain a higher number of cells of the detector strain. One may also infer that droplets containing higher numbers of detector strain cells must also contain producer strain cells which generated higher amounts of L-tryptophan.

[0188] Using the microfluidic system, droplets exhibiting high levels of fluorescence are separated from the remainder of the droplet pool and collected. These droplets are then spread on solid media, which is then incubated to recover variants of the producer strain which exhibit higher production of L-tryptophan. Individual clonal isolates are then analyzed in a secondary screen to confirm increased L-tryptophan production: colonies are inoculated into Luria-Bertani (LB) medium and cultured for several days, and culture supernatants are analyzed for L-tryptophan concentration via high performance liquid chromatography (HPLC).

Example 3

[0189] A strain of E. coli is engineered to overproduce L-tryptophan via replacement of the native trpABCDE promoter with a strong constitutive promoter. However, feedback regulation has been shown to limit the amount of L-tryptophan that can be produced by this engineered E. coli strain. To overcome this feedback regulation and other regulatory phenomena that may limit L-tryptophan production, the engineered strain is subjected to UV-induced random mutagenesis, generating a library of L-tryptophan-producing E. coli strains. Following generation, this library is cultured on solid medium. Prior to plating on a solid medium, the library is sufficiently diluted such that clonal isolates are obtained on solid media following a period of incubation.

[0190] A strain of Saccharomyces cerevisiae that is auxotrophic for L-tryptophan and L-leucine (e.g., W303 and its derivatives) is transformed with a plasmid (named here as pLux) containing the coding sequence of the lux luminescence operon under the control of a strong constitutive promoter (e.g., P.sub.TEF1) as well as the gene or gene operon that allows for intracellular production of L-leucine. Such complementation of the L-leucine auxotroph allows for positive selection of S. cerevisiae cells harboring the pLux plasmid. When cultured in the presence of L-tryptophan but in the absence of L-leucine, the auxotrophic Saccharomyces cerevisiae strain harboring pLux proliferates and generates the machinery necessary to produce luminescence. The proliferation of this strain can be monitored via luminescence measurements, namely by measuring the amount of light emitted by the cells at wavelength or range of wavelengths appropriate for the given lux luminescence operon. This auxotrophic Saccharomyces cerevisiae strain will be referred to hereafter as the detector strain.

[0191] The producer strain library is recovered from solid medium, then diluted and inoculated into a minimal medium (e.g., M9 minimal medium with 4 g/L glucose). This culture is grown for 4-8 hours at 37 C. with shaking at 200 rpm, then diluted to an OD.sub.600 of 0.02 using the same minimal medium. The detector strain is inoculated into a synthetically defined medium containing L-tryptophan (to allow for cell growth) but missing L-leucine (to ensure maintenance of the pLux plasmid). This detector strain culture is grown for 4-8 hours at 30 C. with shaking at 200 rpm. The detector strain culture is then washed with an isotonic buffer and resuspended using a synthetically defined medium missing both L-tryptophan and L-leucine. Microfluidic droplets 20 pL in volume are generated using a microfluidic system in which the aqueous phase comprising the producer strain diluted in minimal medium is separated into droplets by a fluorinated oil (e.g., HFE7500) containing a fluorinated surfactant. These microfluidic droplets are collected and subjected to picoinjection, in which a small, defined volume (5 pL) of detector strain culture is added to each microfluidic droplet, thereby contacting cells of the producer strain with cells of the detector strain within microfluidic droplets. The picoinjected droplets are then collected and incubated at 30 C. to allow for growth of the producer strain, production of L-tryptophan, subsequent growth of the detector strain, and concomitant production of the fluorescent protein.

[0192] The microfluidic droplets are then analyzed using the microfluidic system. The luminescence of each droplet is analyzed by measuring the amount of light emitted by each droplet over a range of wavelengths appropriate for the chosen lux luminescence. Droplets exhibiting higher luminescence must contain higher concentrations of luminescence machinery and must therefore contain a higher number of cells of the detector strain. One may also infer that droplets containing higher numbers of detector strain cells must also contain producer strain cells which generated higher amounts of L-tryptophan.

[0193] Using the microfluidic system, droplets exhibiting high levels of luminescence are separated from the remainder of the droplet pool and collected. These droplets are then spread on solid media, which is then incubated to recover variants of the producer strain which exhibit higher production of L-tryptophan. Individual clonal isolates are then analyzed in a secondary screen to confirm increased L-tryptophan production: colonies are inoculated into Luria-Bertani (LB) medium and cultured for several days, and culture supernatants are analyzed for L-tryptophan concentration via high performance liquid chromatography (HPLC).

Example 4

[0194] To identify novel producers of L-tryptophan, a soil environmental sample is washed with an isotonic buffer to recover bacteria present in the sample. These bacteria are then diluted using a chemically defined medium that does not contain L-tryptophan, generating a library of potential producer strains.

[0195] A strain of Saccharomyces cerevisiae that is auxotrophic for L-tryptophan and L-leucine (e.g., W303 and its derivatives) is transformed with a plasmid (named here as pFluor) containing the coding sequence of a fluorescent protein (e.g., GFP, eGFP, mCherry, RFP, etc.) under the control of a strong constitutive promoter (e.g., P.sub.TEF1) as well as the gene or gene operon that allows for intracellular production of L-leucine. Such complementation of the L-leucine auxotroph allows for positive selection of S. cerevisiae cells harboring the pFluor plasmid. When cultured in the presence of L-tryptophan but in the absence of L-leucine, the auxotrophic Saccharomyces cerevisiae strain harboring pFluor proliferates and expresses the fluorescent protein intracellularly. The proliferation of this strain can be monitored via fluorescence measurements, namely illuminating the cells with light of a wavelength or range of wavelengths and measuring the amount of light emitted by the cells at a wavelength or range of wavelengths greater than the wavelength(s) used for illumination. This auxotrophic Saccharomyces cerevisiae strain will be referred to hereafter as the detector strain.

[0196] The detector strain is inoculated into a synthetically defined medium containing L-tryptophan (to allow for cell growth) but missing L-leucine (to ensure maintenance of the pFluor plasmid. This detector strain culture is grown for 4-8 hours at 30 C. with shaking at 200 rpm. The detector strain culture is then washed with an isotonic buffer and resuspended using a synthetically defined medium missing both L-tryptophan and L-leucine. Microfluidic droplets 20 pL in volume are generated using a microfluidic system in which the aqueous phase comprising the library of producer strains diluted in a chemically defined medium is separated into droplets by a fluorinated oil (e.g., HFE7500) containing a fluorinated surfactant. These microfluidic droplets are collected and subjected to picoinjection, in which a small, defined volume (5 pL) of detector strain culture is added to each microfluidic droplet, thereby contacting cells of the producer strain with cells of the detector strain within microfluidic droplets. The picoinjected droplets are then collected and incubated at 30 C. to allow for growth of the producer strain, production of L-tryptophan, subsequent growth of the detector strain, and concomitant production of the fluorescent protein. The microfluidic droplets are then analyzed using the microfluidic system. The fluorescence of each droplet is analyzed by illuminating the droplet with a laser having a wavelength corresponding to the excitation maximum of the fluorescent protein of interest and measuring the amount of light emitted by the droplet at a range of wavelengths longer than the wavelength used for illumination/excitation. Droplets exhibiting higher fluorescence must contain higher concentrations of fluorescent protein and must therefore contain a higher number of cells of the detector strain. One may also infer that droplets containing higher numbers of detector strain cells must also contain producer strain cells which generated higher amounts of L-tryptophan.

[0197] Using the microfluidic system, droplets exhibiting high levels of fluorescence are separated from the remainder of the droplet pool and collected. These droplets are then spread on solid media, which is then incubated to recover variants of the producer strain which exhibit higher production of L-tryptophan. Individual clonal isolates are then analyzed in a secondary screen to confirm increased L-tryptophan production: colonies are inoculated into Luria-Bertani (LB) medium and cultured for several days, and culture supernatants are analyzed for L-tryptophan concentration via high performance liquid chromatography (HPLC).