초록 ▼

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 ▼

1. A method of screening an environmental library for an agent that modulates interaction of a first test protein linked to a DNA binding moiety and a second test protein linked to a transcriptional activation moiety, comprisingproviding an environmental expression library containing a plurality of

1. A method of screening an environmental library for an agent that modulates interaction of a first test protein linked to a DNA binding moiety and a second test protein linked to a transcriptional activation moiety, comprisingproviding an environmental expression library containing a plurality of recombinant prokaryotic clones, wherein DNA for generating the library is naturally occurring and obtained from a mixed population of organisms;co-encapsulating in a suitable microenvironment at least of the prokaryotic clones and a second recombinant clone expressing a fluorescent protein, with the first test protein and the second test protein; andscreening the microenvironment by fluorescence activated cell sorting (FACS) analysis to determining ability of an agent produced by the prokaryotic clone to modulate interaction of the first test protein linked to a DNA binding moiety with the second test protein covalently linked to a transcriptional activation moiety to produce a change in fluorescence of the fluorescent protein, wherein the change indicates the presence of the agent. 2. The method of claim 1, wherein the agent is an enzyme or small molecule. 3. The method of claim 2, wherein the enzyme is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferase, phosphatase, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, epozide hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases. 4. The method of claim 1, wherein the modulation inhibits the expression of a reporter gene controlled by the first protein or the second protein. 5. The method of claim 1, wherein the modulation inhibits the expression of a reporter gene controlled by the first protein or the second protein. 6. The method of claim 1 , wherein the second recombinant cell is a eukaryotic cell. 7. The method of claim 1, wherein the second recombinant cell is a prokaryotic cell. 8. The method of claim 1, wherein the microenvironment is a liposome, gel microdrop, bead, agarose, cell, ghost red blood cell or ghost macrophage. 9. The method of claim 8, wherein the liposomes are prepared from one or more phospholipids, glycolipids, steroids, alkyl phosphates or fatty acid esters. 10. The method of claim 9, wherein the phospholipids are selected from the group consisting of lecithin, sphingomyelin and dipalmitoyl. 11. The method of claim 9, wherein the steroids are selected from the group consisting of cholesterol, chlorestanol and lanosterol. 12. The method of claim 1, wherein the fluoresent protein is a fluorescent dye, a bioluminescent material, a chemiluminescent material, or a fluoresecent enzymatic substrate. 13. The method of claim 12, wherein the bioluminescent material is green fluorescent protein (GFP) or red fluorescent protein (RFP). 14. The method of claim 1, wherein at least one test protein is derived from a mixed population of organisms. 15. The method of claim 1, wherein the DNA for generation the library is normalized.