Claims
- 1. A method for identifying bioactivities or biomolecules using high throughput screening of genomic DNA comprising:a) providing an expression library containing a plurality of clones, wherein the DNA for generating the library is obtained from a mixed population of organisms; b) encapsulating a bioactive fluorescent substrate and at least one clone of the library in a gel microdroplet, wherein the substrate is fluorescent in the presence of a bioactivity or biomolecule; c) screening the microdroplet with a fluorescent analyzer that detects bioactive fluorescence; and d) identifying clones detected as positive for bioactive fluorescence, wherein fluorescence is indicative of DNA that encodes a bioactivity or biomolecule.
- 2. The method of claim 1, wherein the bioactivity is an enzyme is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, epozide hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.
- 3. The method of claim 1, wherein the library is generated in a prokaryotic cell.
- 4. The method of claim 3, wherein the prokaryotic cell is gram negative.
- 5. The method of claim 4, wherein the prokaryotic cell is E. coli.
- 6. The method of claim 5, wherein prior to step b), the E. coli is transferred to a Streptomyces sp.
- 7. The method of claim 6, wherein the Streptomyces sp. is Streptomyces venezuelae.
- 8. The method of claim 1, wherein the expression library contains DNA obtained from extremophiles.
- 9. The method of claim 8, wherein the extremophiles are thermophiles.
- 10. The method of claim 9 wherein the extremeophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.
- 11. The method of claim 1 wherein the bioactive substrate comprises C12FDG.
- 12. The method of claim 1, wherein the bioactive substrate comprises a lipophilic tail.
- 13. The method of claim 1, wherein the samples are heated before step b).
- 14. The method of claim 11, wherein the heating is at about 70° C.
- 15. The method of claim 14, wherein the heating occurs at about 30 minutes.
- 16. The method of claim 1, wherein the fluorescent analyzer comprises a FACS apparatus.
- 17. The method of claim 1, wherein the expression library is biopanned before step b).
- 18. The method of claim 1, including the additional steps of: subjecting an enzyme encoded by the DNA identified in step d) to directed evolution comprising the steps of:a) subjecting the enzyme to non-directed mutagenesis; and b) screening mutant enzymes produced in step a) for a mutant enzyme.
- 19. The method of claim 1, wherein the prokaryotic expression library is normalized before step b).
- 20. The method of claim 1, further comprising co-encapsulating an indicator cell in step b).
- 21. The method of claim 1, wherein the library is a prokaryotic expression library.
- 22. A method for identifying bioactivities or biomolecules using high throughput screening of prokaryotic genomic DNA in a culture-independent system comprising:a) generating a prokaryotic expression library containing a plurality of clones, wherein the DNA for generating the library is obtained from a mixed population of organisms; b) inserting a bioactive fluorescent substrate into the clones of the library, wherein the substrate is fluorescent in the presence of a bioactivity or biomolecule; c) screening the clones with a fluorescent analyzer that detects bioactive fluorescence; and d) identifying clones detected as positive for bioactive fluorescence, wherein fluorescence is indicative of DNA that encodes a bioactivity or biomolecule.
- 23. The method of claim 22, further comprising encapsulation the clone and the bioactive substrate prior to screening.
Parent Case Info
This application is a continuation-in-part of U.S. application Ser. No. 08/876,276, filed Jun. 16, 1997.
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Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
08/876276 |
Jun 1997 |
US |
Child |
09/098206 |
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US |