Production and monitoring of metabolites in cells
Methods of making and monitoring metabolites in cells are provided.
1. A method of selecting a subset of microbes for the production of a metabolite comprising:
(a) providing a population of microbes,
wherein the population of microbes has been genetically modified to include exogenous DNA encoding for a reporter, the reporter being a fluorescent protein,
wherein the population of microbes has been genetically modified to include exogenous DNA encoding a sensor biomolecule, wherein the sensor biomolecule is a transcription factor, which when expressed regulates expression of the reporter by the microbes,
wherein the population of microbes has been genetically modified to include exogenous DNA encoding genes to produce a metabolite binding partner of the sensor, wherein the sensor biomolecule and the metabolite binding partner is a member pair selected from the group consisting of cdaR/glucaric acid, ttgR/naringennin, tetR/tetracycline derivates, benM/muconic acid, alkS/medium chain n-alkanes, xylR/xylose, araC/Arabinose, gntR/Gluconate, galS/Galactose, trpR/tryptophan, qacR/Berberine, rmrR/Phytoalexin, cymR/Cumate, melR/Melibiose, rafR/Raffinose, nahR/Salicylate, nocR/Nopaline, cicR/Chlorobenzoate, varR/Virginiamycin, PhoR/Phosphate, MalK/Malate, GlnK/Glutamine, Retinoic acid receptor/Retinoic acid, Estrogen receptor/Estrogen and Ecdysone receptor/Ecdysone, and
wherein the microbes produce the metabolite binding partner which binds to the sensor to induce expression of the reporter in a manner dependent on the concentration of the produced metabolite, and
(b) screening the population of microbes by detecting the reporter to identify a subset of microbes.
2. A method of selecting a subset of microbes for the production of a metabolite comprising:
(a) providing a population of microbes,
wherein the population of microbes has been genetically modified to include exogenous DNA encoding for a reporter, the reporter being a fluorescent protein, wherein the reporter is a member selected from the group consisting of mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, T-Sapphire, Firefly (FLuc), modified firefly (Ultra-Clo), Click beetle (CBLuc), Sea pansy (RLuc), Copepod crustacean (GLuc), and Ostracod crustacean (CLuc),
wherein the population of microbes has been genetically modified to include exogenous DNA encoding a sensor biomolecule, wherein the sensor biomolecule is a transcription factor, which when expressed regulates expression of the reporter by the microbes,
wherein the population of microbes has been genetically modified to include exogenous DNA encoding genes to produce a metabolite binding partner of the sensor, wherein the sensor biomolecule and the metabolite binding partner is a member pair selected from the group consisting of cdaR/glucaric acid, ttgR/naringennin, tetR/tetracycline derivates, benM/muconic acid, alkS/medium chain n-alkanes, xylR/xylose, araC/Arabinose, gntR/Gluconate, galS/Galactose, trpR/tryptophan, qacR/Berberine, rmrR/Phytoalexin, cymR/Cumate, melR/Melibiose, rafR/Raffinose, nahR/Salicylate, nocR/Nopaline, cicR/Chlorobenzoate, varR/Virginiamycin, PhoR/Phosphate, MalK/Malate, GlnK/Glutamine, Retinoic acid receptor/Retinoic acid, Estrogen receptor/Estrogen and Ecdysone receptor/Ecdysone, and
wherein the microbes produce the metabolite binding partner which binds to the sensor to induce expression of the reporter in a manner dependent on the concentration of the produced metabolite, and
(b) screening the population of microbes by detecting the reporter to identify a subset of microbes.
3. The method of claim 1 , wherein the screening is carried out by fluorescence activated cell sorting, microscopy, microtiter plate assay, emulsion assay, microfluidic assay, pull-down assay or luciferase high throughput screening.
4. The method of claim 1 , further comprising
genetically modifying the subset of microbes to alter genes that affect production of the metabolite directly or indirectly, and
screening the subset of microbes by detecting the reporter to identify a subsequent subset of microbes.
5. The method of claim 1 , wherein the reporter is enhanced green fluorescent protein (EGFP).
6. The method of claim 1 , wherein the screening further comprises selecting a subset of microbes that produce the metabolite based on detection of the reporter.
7. The method of claim 2 , wherein the screening is carried out by fluorescence activated cell sorting, microscopy, microtiter plate assay, emulsion assay, microfluidic assay, pull-down assay or luciferase high throughput screening.
8. The method of claim 2 , further comprising
genetically modifying the subset of microbes to alter genes that affect production of the metabolite directly or indirectly, and
screening the subset of microbes by detecting the reporter to identify a subsequent subset of microbes.
9. The method of claim 2 , wherein the reporter is EGFP.
10. The method of claim 2 , wherein the screening further comprises selecting a subset of microbes that produce the metabolite based on detection of the reporter.