An efficient gene deletion system for Bacillus thuringiensis

作者：Doruk Tugrul; Gedik Sedef Tunca^*

来源：Biologia, 2013, 68(3): 358-364.

DOI：10.2478/s11756-013-0184-4

• Bacillus thuringiensis
• PCR
• homologous recombination
• genome manipulation
• deletion mutant

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.

• 出版日期2013-6