Format

Send to

Choose Destination
Nat Protoc. 2011 Jul 7;6(8):1085-104. doi: 10.1038/nprot.2011.346.

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

Author information

1
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA.

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 ~10 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.

PMID:
21738123
PMCID:
PMC3564556
DOI:
10.1038/nprot.2011.346
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center