Protein activity screening of clones having DNA from uncultivated microorganisms

Abstract

Disclosed is a process of screening clones having DNA from an uncultivated microorganism for a specified protein, e.g. enzyme, activity by screening for a specified protein, e.g. enzyme, activity in a library of clones prepared by (i) recovering DNA from a DNA population derived from at least one uncultivated microorganism; and (ii) transforming a host with recovered DNA to produce a library of clones which is screened for the specified protein, e.g. enzyme, activity.

Description

RELATED APPLICATIONS [0001] This application is a continuation application of U.S. patent application Ser. No. 09 / 713,176, filed on Nov. 14, 2000, which is a continuation application of U.S. patent application Ser. No. 08 / 988,224, filed Dec. 10, 1997, issued as U.S. Pat. No. 6,280,926, which is a divisional application of U.S. patent application Ser. No. 08 / 657,409, filed on Jun. 3, 1996, issued as U.S. Pat. No. 5,958,672, which is a continuation-in-part of U.S. application Ser. No. 08 / 568,994, filed on Dec. 7, 1995, now abandoned, which was a continuation-in-part of U.S. application Ser. No. 08 / 503,606, filed on Jul. 18, 1995, issued as U.S. Pat. No. 6,004,788. All of the disclosures of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION [0002] This invention relates to the field of preparing and screening libraries of clones containing microbially derived DNA. BACKGROUND OF THE INVENTION [0003] Naturally occurring assemblages of microorganisms often...

AI Technical Summary

Benefits of technology

[0010] The f-factor (or fertility factor) in E. cold is a plasmid which effects high frequency transfer of itself during conjugation and less frequent transfer of the bacterial chromosome itself. To achieve and stably propagate large DNA fragments from mixed microbial samples, a particularly preferred embodiment is to use a cloning vector containing an f-factor origin of replication to generate genomic libraries that can be replicated with a high degree of fidelity. When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable “environmental DNA library.”

[0026] The ability to select and combine desired components from a library of polyketides and postpolyketide biosynthesis genes for generation of novel polyketides for study is appealing. The method(s) of the present invention make it possible to and facilitate the cloning of novel polyketide syntheses, since one can generate gene banks with clones containing large inserts (especially when using the f-factor based vectors), which facilitates cloning of gene clusters.

[0045] Preferably the probe DNA is “labeled” with one partner of a specific binding pair (i.e. a ligand) and the other partner of the pair is bound to a solid matrix to provide ease of separation of target from its source. The ligand and specific binding partner can be selected from, in either orientation, the following: (1) an antigen or hapten and an antibody or specific binding fragment thereof; (2) biotin or iminobiotin and avidin or streptavidin; (3) a sugar and a lectin specific therefor; (4) a protein, e.g. enzyme, and an inhibitor therefor; (5) an apoenzyrne and cofactor; (6) complementary homopolymeric oligonucleotides; and (7) a hormone and a receptor therefor. The solid phase is preferably selected from: (1) a glass or polymeric surface; (2) a packed column of polymeric beads; and (3) magnetic or paramagnetic particles.

[0054] A particularly preferred type of vector for use in the present invention contains an f-factor origin of replication. The f-factor (or fertility factor) in E. coli is a plasmid which effects high frequency transfer of itself during conjugation and less frequent transfer of the bacterial chromosome itself. A particularly preferred embodiment is to use cloning vectors, referred to as “fosmids” or bacterial artificial chromosome (BAC) vectors. These are derived from the E. cold f-factor and are able to stably integrate large segments of genomic DNA. When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable “environmental DNA library.”

Problems solved by technology

It has been suggested that a large fraction of this diversity thus far has been unrecognized due to difficulties in enriching and isolating microorganisms in pure culture.

Therefore, it has been difficult or impossible to identify or isolate valuable proteins, e.g. enzymes, from these samples.

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