Methylotroph cloning vehicle

Details Methylotroph cloning vehicle Methylotroph cloning vehicle

C - Chemistry, Metallurgy

12

N

195/1.16, 195/1.

C12N 15/74 (2006.01) C12N 1/21 (2006.01) C12P 13/04 (2006.01)

Patent

CA 1335576

A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C1-utilizing host and in a C1-utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C1-utilizing host to the C1-utilizing host: DNA providing resistance to two antibiotics to which the wild-type C1-utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C1-utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C1-utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C1-utilizing (e. g., E. coli) host, and then conjugated with a selected C1-utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C1 gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields.

490736

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