Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1985-01-03
1987-12-15
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
4351721, 935 11, C12N 1500
Patent
active
047133376
ABSTRACT:
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.
REFERENCES:
Kleckner et al., J. Mol. Biol., 116:125-159, 1977.
Scherer et al., PNAS (USA), 76(10):4951-4955, 1979.
Jasin et al., J. Bacteriol., 159(2):783-786, 1984.
Clark, Ann. Rev. Genet., 7:67-86, 1974.
Miller, J., in Experiments in Molecular Genetics, Cold Spring Harbor, pp. 196-200, 1972.
"Replacement and Amplification of Bacterial Genes with Sequences Altered in Vitro" by N. I. Gutterson et al., Proc. Natl. Acad. Sci., vol. 80, pp. 4894-4898 (1983).
"Use of Cloned mtl Genes of Escherichia coli to Introduce mtl Deletion Mutations into the Chromosome" by C. A. Lee et al., J. Bact., vol. 153, pp. 685-692 (1983).
"[12] One-Step Gene Disruption in Yeast" by R. J. Rothstein, Methods in Enzymology, vol. 101, pp. 202-211, (1983).
Jasin Maria
Schimmel Paul R.
Massachusetts Institute of Technology
Mays Thomas D.
Pabst Patrea L.
Wiseman Thomas G.
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