Dergi makalesi Açık Erişim
Doruk, Tugrul; Gedik, Sedef Tunca
It is not easy to manipulate biosynthetic genes of Bacillus thuringiensis since there is a powerful methyl-specific restriction system in this microorganism. In this study, a PCR-based system was used to delete polyphosphate kinase gene (ppk) of Bacillus thuringiensis israelensis (Bti) by replacing the wild-type gene with a cassette containing the apramycin resistance gene as selectable marker. lambda-Red was used to promote recombination in Escherichia coli between a PCR-amplified apramycin resistance cassette (linear deletion cassette selectable in E. coli and Bti) and Bti DNA on a plasmid. The isolated mutant plasmid was transferred to Bti by conjugation. Double cross-over transformants were screened for their antibiotic resistance and the mutation was proven by PCR, southern blot hybridization and RT-PCR. The described method, which uses the advantage of quick plasmid construction in E. coli and simple transformation of linear deletion cassette, is very useful to delete entire gene/genes of Bti without any polar effects on genes transcriptionally downstream.
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bib-6a1e474a-905f-4a15-b90c-090c41df1c06.txt
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