Complementation of Burkholderia pseudomallei Strains

Deletion strains were complemented with a bpeT or bpeEF-oprC gene(s) originating from strain 1026b or Bp82 utilizing the mini-Tn7 system, which allows stable and site-specific single-copy insertions into the B. pseudomallei genome at three possible glmS-associated attTn7 sites (50 (link)). The respective mini-Tn7 elements, along with an empty-mini-Tn7 element used as a control, were transferred to the target B. pseudomallei strains either via conjugation from E. coli or by electroporation, and glmS-associated insertions were verified as previously described (50 (link), 51 (link)). Mini-Tn7 insertions at the glmS2-associated attTn7 site were routinely retained for further studies, unless noted otherwise. The inducible E. coli trp/lac operon hybrid P_tac promoter was used for regulated expression of the bpeEF-oprC genes. BpeEF-OprC expression was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Gold Biotechnology, St. Louis, MO) (28 (link)).

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Podnecky N.L., Rhodes K.A., Mima T., Drew H.R., Chirakul S., Wuthiekanun V., Schupp J.M., Sarovich D.S., Currie B.J., Keim P, & Schweizer H.P. (2017). Mechanisms of Resistance to Folate Pathway Inhibitors in Burkholderia pseudomallei: Deviation from the Norm. mBio, 8(5), e01357-17.

Publication 2017

isopropyl-β-d-thiogalactopyranoside (IPTG;

Deletion E coli Electroporation Expression genes Gene Genome Glms Gold Hybrid Insertions Iptg Lac operon Ptac Strains

Corresponding Organization :

Other organizations : Colorado State University, University of Florida, Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Translational Genomics Research Institute, Menzies School of Health Research, University of the Sunshine Coast, Northern Arizona University

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Variable analysis

independent variables

Complementation of deletion strains with a bpeT or bpeEF-oprC gene(s) originating from strain 1026b or Bp82 utilizing the mini-Tn7 system
Induction of BpeEF-OprC expression by addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG)

dependent variables

Not explicitly mentioned

control variables

Stable and site-specific single-copy insertions of mini-Tn7 elements into the B. pseudomallei genome at three possible glmS-associated attTn7 sites
Retention of mini-Tn7 insertions at the glmS2-associated attTn7 site for further studies, unless noted otherwise

controls

Empty-mini-Tn7 element used as a negative control

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