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Method for deletion of a gene from a bacteria

Jasin; Maria ; Schimmel; Paul R

15 December 1987

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  • Titolo:
    Method for deletion of a gene from a bacteria
  • Autore: Jasin; Maria ; Schimmel; Paul R
  • Anno: 15 December 1987
  • Descrizione: Disclosed is a method for the deletion of a gene from a bacteria using a single step procedure that is applicable to any essential or nonessential gene which has been cloned. The process requires the construction of chromosomal deletions by transformation of a cell strain with linear DNA fragments containing a locus for resistance to an antibiotic, or any other gene allowing for rapid phenotypic selection, flanked by sequences homologous to a closely spaced region on the cell chromosome. By selecting for a double-crossover event between the homologous sequences, shown by the antibiotic resistant or other detectable phenotype, a chromosome disruption can be selected for which has effectively deleted an entire gene. If the gene is essential to viability, the bacteria may be transformed with a plasmid which has a temperature-sensitive replicon and a wild-type allele of the essential gene. When E. coli is used as the host strain, it is preferable to use a cell strain which carries the recBC and sbcB mutant alleles which inactivate exonucleases which degrade linear DNA fragments. The sbcB mutation supresses the Rec- phenotype of recBC cells so that homologous recombination functions via an alternative pathway. This plasmid maintains cell viability when the chromosomal copy of the desired gene has been deleted, but the resulting cells have a temperature-sensitive phenotype to provide a means for eliminating the plasmid. A key feature of the present invention utilizing extrachromasomal genetic material to maintain the desired phenotype once a gene has been deleted is that the bacteria have a RecA+ phenotype or its equivalent at the time of transformation with the linear DNA fragments, which is immediately changed to a RecA- phenotype once recombination of the fragments has occurred to prevent recombination of the extrachromosomal genetic material with homologous sequences on the chromosome.
  • Soggetti: Chemistry ; Metallurgy ; Biochemistry ; Beer ; Spirits ; Wine ; Vinegar ; Microbiology ; Enzymology ; Mutation OR Genetic Engineering ; Microorganisms OR Enzymes ; Compositions Thereof ; Propagating, Preserving OR Maintaining Microorganisms ; Mutation OR Genetic Engineering ; Culture Media ; Medicine ; Sciences ; Chemistry ; Chemistry
  • Lingua: Inglese
  • Fonte: Espacenet (European Patent Office)

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