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Title: Methylotroph cloning vehicle

Abstract

A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C.sub.1 -utilizing host and in a C.sub.1 -utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C.sub.1 -utilizing host to the C.sub.1 -utilizing host; DNA providing resistance to two antibiotics to which the wild-type C.sub.1 -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 C.sub.1 -utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C.sub.1 -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-C.sub.1 -utilizing (e.g., E. coli) host, and then conjugated with a selected C.sub.1 -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 C.sub.1 gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosyntheticmore » pathway to a desired product, thereby producing an organism that makes the desired product in higher yields.« less

Inventors:
 [1];  [2]
  1. (Deephaven, MN)
  2. (Excelsior, MN)
Issue Date:
Research Org.:
University of Minnesota
OSTI Identifier:
866931
Patent Number(s):
4824786
Application Number:
07/107,244
Assignee:
Regents of University of Minnesota (Minneapolis, MN) ANL
DOE Contract Number:  
AC02-82ER12029
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
methylotroph; cloning; vehicle; comprising; replication; determinant; effective; replicating; non-c; -utilizing; host; dna; allow; mobilized; providing; resistance; antibiotics; wild-type; susceptible; antibiotic; markers; recognition; site; restriction; endonuclease; cos; means; preventing; complementation; mapping; follows; organism; inserted; nuclease; inactivating; marker; form; cosmid; structure; infect; coli; conjugated; selected; mutant; conjugation; phenotypical; changes; loss; auxotrophic; trait; attributed; vector; inactivate; genes; protein; products; catalyze; reactions; divert; compounds; biosynthetic; pathway; desired; product; producing; makes; yields; -utilizing host; cloning vehicle; desired product; vehicle comprising; restriction endonuclease; nuclease recognition; /435/999/

Citation Formats

Hanson, Richard S., and Allen, Larry N. Methylotroph cloning vehicle. United States: N. p., 1989. Web.
Hanson, Richard S., & Allen, Larry N. Methylotroph cloning vehicle. United States.
Hanson, Richard S., and Allen, Larry N. Tue . "Methylotroph cloning vehicle". United States. https://www.osti.gov/servlets/purl/866931.
@article{osti_866931,
title = {Methylotroph cloning vehicle},
author = {Hanson, Richard S. and Allen, Larry N.},
abstractNote = {A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C.sub.1 -utilizing host and in a C.sub.1 -utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C.sub.1 -utilizing host to the C.sub.1 -utilizing host; DNA providing resistance to two antibiotics to which the wild-type C.sub.1 -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 C.sub.1 -utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C.sub.1 -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-C.sub.1 -utilizing (e.g., E. coli) host, and then conjugated with a selected C.sub.1 -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 C.sub.1 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.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1989},
month = {4}
}

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