Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei

Yun Kang, Michael H. Norris, Bruce A. Wilcox, Apichai Tuanyok, Paul S Keim, Tung T. Hoang

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Phage λ-Red proteins are powerful tools for pulling and knocking out chromosomal fragments but have been limited to the γ-proteobacteria. Procedures are described here to easily knock out (KO) and pull out (PO) chromosomal DNA fragments from naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. This system takes advantage of published compliant counterselectable and selectable markers (sacB, pheS, gat and the arabinose-utilization operon) and λ-Red mutant proteins. pheS-gat (KO) or oriT-ColE1ori-gat-ori1600-rep (PO) PCR fragments are generated with flanking 40- to 45-bp homologies to targeted regions incorporated on PCR primers. One-step recombination is achieved by incubation of the PCR product with cells expressing λ-Red proteins and subsequent selection on glyphosate-containing medium. This procedure takes ĝ̂1/410 d and is advantageous over previously published protocols: (i) smaller PCR products reduce primer numbers and amplification steps, (ii) PO fragments suitable for downstream manipulation in Escherichia coli are obtained and (iii) chromosomal KO increases flexibility for downstream processing.

Original languageEnglish (US)
Pages (from-to)1085-1104
Number of pages20
JournalNature Protocols
Volume6
Issue number8
DOIs
StatePublished - Aug 2011
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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