Abstract
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. λ-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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Abbreviations
- aac(3)IV :
-
aminoglycoside 3-N-acetyltransferase gene
- Ap:
-
ampicilin
- Apr:
-
apramycin
- BHI:
-
brain heart infusion
- Bti :
-
Bacillus thuringiensis subsp. israelensis
- Chl:
-
chloramphenicol
- Er:
-
erythromycin
- ermB :
-
rRNA adenine N-6-methyltransferase gene
- Kn:
-
kanamycin
- LB:
-
Luria Bertani
- ppk :
-
polyphosphate kinase gene
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Doruk, T., Gedik, S.T. An efficient gene deletion system for Bacillus thuringiensis . Biologia 68, 358–364 (2013). https://doi.org/10.2478/s11756-013-0184-4
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DOI: https://doi.org/10.2478/s11756-013-0184-4