IPC분류정보
국가/구분 |
United States(US) Patent
등록
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국제특허분류(IPC7판) |
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출원번호 |
US-0848095
(2001-05-03)
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발명자
/ 주소 |
- Short, Jay M.
- Keller, Martin
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출원인 / 주소 |
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대리인 / 주소 |
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인용정보 |
피인용 횟수 :
2 인용 특허 :
11 |
초록
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Disclosed is a process for identifying clones having a specified activity of interest, which process comprises (i) generating one or more expression libraries derived from nucleic acid directly isolated from the environment; and (ii) screening said libraries utilizing a fluorescence activated cell s
Disclosed is a process for identifying clones having a specified activity of interest, which process comprises (i) generating one or more expression libraries derived from nucleic acid directly isolated from the environment; and (ii) screening said libraries utilizing a fluorescence activated cell sorter to identify said clones. More particularly, this is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; (ii) exposing said libraries to a particular substrate or substrates of interest; and (iii) screening said exposed libraries utilizing a fluorescence activated cell sorter to identify clones which react with the substrate or substrates. Also provided is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; and (ii) screening said exposed libraries utilizing an assay requiring co-encapsulation, a binding event or the covalent modification of a target, and a fluorescence activated cell sorter to identify positive clones.
대표청구항
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1. A method for identifying bioactivities or biomolecules using high throughput screening of nucleic acid comprising:a) generating a normalized environmental gene library containing a plurality of clones in E. coli, wherein the nucleic acid for generating the library is naturally occurring and obtai
1. A method for identifying bioactivities or biomolecules using high throughput screening of nucleic acid comprising:a) generating a normalized environmental gene library containing a plurality of clones in E. coli, wherein the nucleic acid for generating the library is naturally occurring and obtained from a mixed population of uncultured organisms; b) transferring a plurality of the clones to myceliate bacteria or myceliate fungi; c) encapsulating a bioactive substrate and at least one clone transferred in b) in a gel microdroplet, wherein a bioactivity or biomolecule produced by the clone is detectable by a change in fluorescence of the substrate prior to contacting with the at least one clone as compared to after the contacting; and d) screening the microdroplet with an assay or an analyzer that detects the presence therein of the change in fluorescence of the substrate, wherein the change indicates the identity of the bioactivity or biomolecule. 2. The method of claim 1, wherein the bioactivity is provided by an enzyme that is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidazes, lignin peroxidases, diarylpropane peroxidazes, epozide hydrolazes, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.3. The method of claim 1, wherein the gene library is an expression library.4. The method of claim 3, wherein the expression library contains DNA obtained from extremophiles.5. The method of claim 4, wherein the extremophiles are thermophiles.6. The method of claim 5, wherein the extremophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.7. The method of claim 1, wherein the bioactive substrate comprises C12FDG.8. The method of claim 1, wherein the bioactive substrate comprises a lipophilic tail.9. The method of claim 1, wherein the clones are heated before step c).10. The method of claim 9, wherein the heating is at about 70° C.11. The method of claim 10, wherein the heating occurs for about 30 minutes.12. The method of claim 1, wherein the analyzer comprises a fluorescent analyzer.13. The method of claim 12, wherein the fluorescent analyzer is a FACS apparatus.14. The method of claim 1, wherein the library is biopanned before step c).15. The method of claim 1, wherein the myceliate bacteria is a Streptomyces sp.16. The method of claim 15, wherein the Sfreptomyces sp. is Streptomyces venezuelae. 17. The method of claim 1, further comprising co-encapsulating an indicator cell in step c).18. The method of claim 1, wherein the analyzer is a chromogenic analyzer.19. The method of claim 1, wherein the assay is an immunoassay.20. A method for identifying bioactivities or biomolecules using high throughput screening of nucleic acid comprising:a) generating a normalized environmental gene library containing a plurality of clones in E.coli, wherein the nucleic acid for generating the library is naturally occurring and obtained from a mixed population of organisms; b) transferring a plurality of the clones to myceliate bacteria or myceliate fungi; c) inserting a polynucleotide into the clones transferred in b), wherein the polynucleotide encodes a bioactive protein substrate, wherein a fluorescence change in the substrate is detectable in the presence of a bioactivity or biomolecule; and d) screening the clones with an assay or an analyzer that detects the presence therein of the fluorescence change in the substrate, wherein the fluorescence change in the substrate identifies the bioactivity or a biomolecule. 21. The method of claim 20, further comprising encapsulating the clone and the bioactive substrate prior to screening.22. The method of claim 21, wherein the bioactivity is provided by an enzyme that is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, hailoperoxidases, lignin peroxidases, diarylpropane peroxidases, epozide hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.23. The method of claim 21, wherein the gene library is an expression library.24. The method of claim 23, wherein the expression library contains DNA obtained from extremophiles.25. The method of claim 24, wherein the extremophiles are thermophiles.26. The method of claim 25, wherein the extremophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.27. The method of claim 21, wherein the bioactive substrate comprises C12FDG.28. The method of claim 21, wherein the bioactive substrate comprises a lipophilic tail.29. The method of claim 21, wherein the clones are heated before step c).30. The method of claim 29, wherein the heating is at about 70° C.31. The method of claim 30, wherein the heating occurs for about 30 minutes.32. The method of claim 21, wherein the analyzer comprises a fluorescent analyzer.33. The method of claim 32, wherein the fluorescent analyzer is a FACS apparatus.34. The method of claim 21, wherein the library is biospanned before step b).35. The method of claim 1, wherein the myceliate fungi is an Actinomyces sp.36. The method of claim 1, wherein the myceliate bacteria is a Streptomyces sp.37. The method of claim 35, wherein the Streptomyces sp. is Streptomyces venezuelae. 38. The method of claim 21, further comprising co-encapsulating an indicator cell in step c).39. The method of claim 21, wherein the analyzer is a chromogenic analyzer.40. The method of claim 21, wherein the assay is an immunoassay.41. The method of claim 36, wherein the bioactive substrate is a fusion protein comprising a protein substrate flanked by two fluorescent proteins that upon contact cause a change in fluorescent signal from the clone, and wherein the effect of the presence of the biomolecule or bioactivity is to cause such contact.42. The method of claim 41, wherein the substrate is for a thioesterase.
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