Journal
PLASMID
Volume 89, Issue -, Pages 49-56Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.plasmid.2016.11.002
Keywords
Marked mutation; Gene inactivation; Burkholderia cepacia complex; Suicide vector; Antibiotic-resistance
Categories
Funding
- BBSRC [BB/MO03531/1]
- University of Umm Al-qura
- Biotechnology and Biological Sciences Research Council [BB/M003531/1] Funding Source: researchfish
- BBSRC [BB/M003531/1] Funding Source: UKRI
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To elucidate the function of a gene in bacteria it is vital that targeted gene inactivation (allelic replacement) can be achieved. Allelic replacement is often carried out by disruption of the gene of interest by insertion of an antibiotic-resistance marker followed by subsequent transfer of the mutant allele to the genome of the host organism in place of the wild-type gene. However, due to their intrinsic resistance to many antibiotics only selected antibiotic-resistance markers can be used in members of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc). Here we describe the construction of improved antibiotic-resistance cassettes that specify resistance to kanamycin, chloramphenicol or trimethoprim effectively in the Bcc and related species. These were then used in combination with and/or to construct a series enhanced suicide vectors, pSHAFT2, pSHAFT3 and pSHAFT-GFP to facilitate effective allelic replacement in the Bcc. Validation of these improved suicide vectors was demonstrated by the genetic inactivation of selected genes in the Bcc species Burkholderia cenocepacia and B. lata, and in the non-Bcc species, B. thailandensis. (C) 2016 The Authors. Published by Elsevier Inc.
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