Claims
- 1. A method for identifying a bioactivity or a biomolecule of interest, comprising:
(a) contacting a library containing a plurality of clones comprising polynucleotides derived from a mixed population of organisms or more than one organism, with at least one oligonucleotide probe labeled with a detectable molecule; and (b) separating clones with an analyzer that detects the detectable molecule.
- 2. The method of claim 1 further comprising:
(a) contacting the separated clones with a reporter system that identifies a polynucleotide encoding a bioactivity or biomolecule of interest; and (b) identifying clones capable of modulating expression or activity of the reporter system thereby identifying a polynucleotide of interest.
- 3. The method of claim 1, wherein the library is an expression library.
- 4. The method of claim 1, wherein the detectable molecule is a fluorescent molecule.
- 5. The method of claim 1, wherein the detectable molecule is a magnetic molecule.
- 6. The method of claim 1, wherein the detectable molecule modulates a magnetic field.
- 7. The method of claim 1, wherein the detectable molecule modulates the dielectric signature of the clone.
- 8. The method of claim 1, wherein the analyzer is a FACS analyzer.
- 9. The method of claim 1, wherein the analyzer is a magnetic field sensing device.
- 10. The method of claim 9, wherein the magnetic field sensing device is a Super Conducting Quantum Interference Device.
- 11. The method of claim 1, wherein the analyzer is a multipole coupling spectroscopy device.
- 12. The method of claim 1, wherein the mixed population of organisms is from an environmental sample.
- 13. The method of claim 1, wherein the mixed population of organisms comprises microorganisms.
- 14. The method of claim 12, wherein the environmental sample contains extremophiles.
- 15. The method of claim 14, wherein the extremophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.
- 16. The method of claim 2, wherein the reporter system is a bioactive substrate.
- 17. The method of claim 16, wherein the bioactive substrate comprises C12FDG.
- 18. The method of claim 17, wherein the bioactive substrate further comprises a lipophilic tail.
- 19. The method of claim 1, further comprising prior to (a):
(i) obtaining polynucleotides from a mixed population of organisms; and (ii) generating a polynucleotide library.
- 20. The method of claim 19, further comprising normalizing the polynucleotides prior to generating the library.
- 21. The method of claim 1, wherein the clones are encapsulated in a microenvironment.
- 22. The method of claim 21, wherein the microenvironment is selected from beads, high temperature agaroses, gel microdroplets, cells, ghost red blood cells, macrophages, or liposomes.
- 23. The method of claim 22, wherein the clones are encapsulated in a gel microdroplet.
- 24. The method of claim 1, wherein the polynucleotide of interest encodes an enzyme.
- 25. The method of claim 24, wherein the enzyme is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, eposize hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.
- 26. The method of claim 1, wherein the reporter system comprises a detectable label.
- 27. The method of claim 1, wherein the reporter system comprises a first test protein linked to a DNA binding moiety and a second test protein linked to a transcriptional activation moiety, wherein modulation of the interaction of the first test protein linked to a DNA binding moiety with the second test protein linked to a transcription activation moiety results in a change in the expression of a detectable protein.
- 28. The method of claim 1, wherein the polynucleotide of interest encodes a small molecule.
- 29. The method of claim 1, wherein the polynucleotide of interest, or fragments thereof, comprise one or more operons, or portions thereof.
- 30. The method of claim 29, wherein the operons, or portions thereof, encodes a complete or partial metabolic pathway.
- 31. The method of claim 30, wherein the operons or portions thereof encoding a complete or partial metabolic pathway encodes polyketide syntheses.
- 32. A method for identifying a polynucleotide encoding a polypeptide of interest comprising:
co-encapsulating in a microenvironment a plurality of library clones containing DNA obtained from a mixed population of organisms, with a mixture of oligonucleotide probes comprising a detectable label and at least a portion of a polynucleotide sequence encoding a polypeptide of interest having a specified bioactivity under such conditions and for such time as to allow interaction of complementary sequences; and identifying clones containing a complement to the oligonucleotide probe encoding the polypeptide of interest by separating clones with an analyzer that detects the detectable label.
- 33. A method for high throughput screening of a polynucleotide library for a polynucleotide of interest that encodes a molecule of interest, comprising:
(a) contacting a library containing a plurality of clones comprising polynucleotides derived from a mixed population of organisms with a plurality of oligonucleotide probes labeled with a detectable molecule; and (b) separating clones with an analyzer that detect the detectable molecule.
- 34. The method of claim 33, further comprising:
(a) contacting the separated clones with a reporter system that identifies a polynucleotide encoding the molecule of interest; and (b) identifying clones capable of modulating expression or activity of the reporter system thereby identifying a polynucleotide of interest.
- 35. The method of claim 33, wherein the library is an expression library.
- 36. The method of claim 33, wherein the mixed population of organisms is from an environmental sample.
- 37. The method of claim 33, wherein the mixed population of organisms comprises microorganisms.
- 38. The method of claim 36, wherein the environmental sample contains extremophiles.
- 39. The method of claim 38, wherein the extremophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.
- 40. The method of claim 34, wherein the reporter system is a bioactive substrate.
- 41. The method of claim 40, wherein the bioactive substrate comprises C12FDG.
- 42. The method of claim 41, wherein the bioactive substrate further comprises a lipophilic tail.
- 43. The method of claim 33, further comprising prior to (a):
(i) obtaining polynucleotides from a mixed population of organisms; and (ii) generating a polynucleotide library.
- 44. The method of claim 43, further comprising normalizing the polynucleotides prior to generating the library.
- 45. The method of claim 33, wherein the clones are encapsulated in a gel microdrop.
- 46. The method of claim 33, wherein the polynucleotide of interest encodes an enzyme.
- 47. The method of claim 46, wherein the enzyme is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, eposize hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.
- 48. The method of claim 34, wherein the reporter system comprises a detectable label.
- 49. The method of claim 33, wherein the reporter system comprises a first test protein linked to a DNA binding moiety and a second test protein linked to a transcriptional activation moiety, wherein modulation of the interaction of the first test protein linked to a DNA binding moiety with the second test protein linked to a transcription activation moiety results in a change in the expression of a detectable protein.
- 50. The method of claim 33, wherein the polynucleotide of interest encodes a small molecule.
- 51. The method of claim 33, wherein the polynucleotide of interest, or fragments thereof, comprise one or more operons, or portions thereof.
- 52. The method of claim 51, wherein the operons, or portions thereof, encodes a complete or partial metabolic pathway.
- 53. The method of claim 52, wherein the operons or portions thereof encoding a complete or partial metabolic pathway encodes polyketide syntheses.
- 54. The method of claim 33, wherein the analyzer is a fluorescence activated cell sorting (FACS) apparatus.
- 55. The method of claim 33, wherein the analyzer is a magnetic field sensing device.
- 56. The method of claim 55, wherein the magnetic field sensing device is a Super Conducting Quantum Interference Device.
- 57. The method of claim 33, wherein the analyzer is a multipole coupling spectroscopy device.
- 58. The method of claim 33, wherein the plurality of oligonucleotide probes have different nucleic acid sequences.
- 59. The method of claim 58, wherein the sequences are portions of a polynucleotide encoding a molecule of interest.
- 60. The method of claim 33, wherein the plurality of oligonucleotide probes have the same nucleic acid sequence.
- 61. A method of screening for a polynucleotide encoding an activity of interest, comprising:
(a) obtaining polynucleotides from an environmental sample; (b) normalizing the polynucleotides obtained from the sample; (c) generating a library from the normalized polynucleotides; (d) contacting the library with a plurality of oligonucleotide probes comprising a detectable label and at least a portion of a polynucleotide sequence encoding a polypeptide of interest having a specified activity to select library clones positive for a sequence of interest; and (e) selecting clones with an analyzer that detects the detectable label.
- 62. The method of claim 61, further comprising:
(a) contacting the selected clones with a reporter system that identifies a polynucleotide encoding the activity of interest; and (b) identifying clones capable of modulating expression or activity of the reporter system thereby identifying a polynucleotide of interest; wherein the positive clones contain a polynucleotide sequence encoding an activity of interest which is capable of catalyzing the bioactive substrate.
- 63. A method for screening polynucleotides, comprising:
(a) contacting a library of polynucleotides wherein the polynucleotides are derived from a mixed population of organism with a probe oligonucleotide labeled with a fluorescence molecule, which fluoresce upon binding of the probe to a target polynucleotide of the library, to select library polynucleotides positive for a sequence of interest; (b) separating library members that are positive for the sequence of interest with a fluorescent analyzer that detects fluorescence; and (c) expressing the selected polynucleotides to obtain polypeptides.
- 64. The method of claim 63, further comprising:
(a) contacting the polypeptides with a reporter system; and (b) identifying polynucleotides encoding polypeptides capable of modulating expression or activity of the reporter system.
- 65. A method for obtaining an organism from a mixed population of organisms in a sample comprising:
(a) encapsulating in a microenvironment at least one organism from the sample; (b) incubating the encapsulated at least one organism under such conditions and for such a time to allow the at least one microorganism to grow or proliferate; and (c) sorting the encapsulated at least one organism by a flow cytometer to obtain an organism from the sample.
- 66. The method of claim 65, wherein the mixed population of organisms is from an environmental sample.
- 67. The method of claim 65, wherein the mixed population of organisms comprises microorganisms.
- 68. The method of claim 66, wherein the environmental sample contains extremophiles.
- 69. The method of claim 68, wherein the extremophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.
- 70. The method of claim 65, wherein the flow cytometer comprises a magnetic field sensing device.
- 71. The method of claim 70, wherein the magnetic field sensing device is a Super Conducting Quantum Interference Device.
- 72. The method of claim 65, wherein the flow cytometer is a multipole coupling spectroscopy device.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 09/685,432, filed Oct. 10, 2000 (pending); which is a continuation-in-part of U.S. patent application Ser. No. 09/444,112, filed Nov. 22, 1999 (pending); which is a continuation-in-part of U.S. patent application Ser. No. 09/098,206, filed Jun. 16, 1998 (pending), which application is a continuation-in-part of U.S. patent application Ser. No. 08/876,276, filed Jun. 16, 1997 (pending), the contents of which are all incorporated by reference in their entirety herein.
Continuation in Parts (4)
|
Number |
Date |
Country |
Parent |
09685432 |
Oct 2000 |
US |
Child |
09738871 |
Dec 2000 |
US |
Parent |
09444112 |
Nov 1999 |
US |
Child |
09685432 |
Oct 2000 |
US |
Parent |
09098206 |
Jun 1998 |
US |
Child |
09444112 |
Nov 1999 |
US |
Parent |
08876276 |
Jun 1997 |
US |
Child |
09098206 |
Jun 1998 |
US |