Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria.
The effects of electroporation temperature, biochemical pretreatment of cells and stage of culture on electroporation efficiency for slow-growing mycobacteria were investigated. The efficiency of transformation into Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium intracellulare increased markedly with temperature. In contrast, the efficiency of transformation into Mycobacterium smegmatis, a fast-growing species, was higher at 0 degree C and decreased with temperature. While stage of culture had little effect, a further increase in efficiency of 2-4-fold was obtained following glycine or ethionamide pretreatment. Electroporation at 37 degrees C has been chosen as a standard condition for slow-growing species as it usually resulted in a transformation efficiency several orders of magnitude higher than that obtained at 0 degree C.
Wards
B J
BJ
AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand. wardsb@agresearch.cri.nz
Collins
D M
DM
D001431:Bacteriological Techniques; D003360:Cosmids; D004269:DNA, Bacterial / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D006358:Hot Temperature; D009161:Mycobacterium / Q000235:genetics* / Q000254:growth & development; D015269:Mycobacterium avium Complex / Q000235:genetics / Q000254:growth & development; D009163:Mycobacterium bovis / Q000235:genetics / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000254:growth & development; D010957:Plasmids
D004269:DNA, Bacterial