Disruption of targeted gene in bacterial chromosome by using a temperature-sensitive plasmid.
The temperature-sensitive plasmid, pSAK3, was used in tryptophan-producing strains for tryptophanase gene disruption to block the degradation of tryptophan. Plasmid pSAK3, which consisted of the pSC103 plasmid containing a tetracycline resistant gene and a disrupted tryptophanase gene inserted by a kanamycin resistant gene, was integrated into the homologous site on the chromosome by gene recombination. Through raising the temperature of cultivation to 42 degrees C and double antibiotics screening process, the strains with disrupted tryptophanase genes could be easily selected and resulted in the generation of stable mutants that no longer exist activity of tryptophanase. Once the strain with a defected tryptophanase gene was obtained, the infection of phage P1 was applied to specifically disrupt the tryptophanase gene of any other strains. This approach also can be applied to disrupt the other targeted genes as an alternative to the conventional mutagenesis.
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Microbiology Division, Development Center for Biotechnology, Taipei, Taiwan, Republic of China.
D001483:Base Sequence; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011815:R Factors; D015183:Restriction Mapping; D013696:Temperature; D013753:Tetracycline Resistance / Q000235:genetics; D014364:Tryptophan / Q000378:metabolism*; D014368:Tryptophanase / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D014364:Tryptophan; D014368:Tryptophanase