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An efficient gene deletion system for Bacillus thuringiensis

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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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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Gebze Institute of Technology, Gebze, Kocaeli, Turkey

    Tugrul Doruk & Sedef Tunca Gedik

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  1. Tugrul Doruk
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  2. Sedef Tunca Gedik
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Correspondence to Sedef Tunca Gedik.

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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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