Expression of beta-lactamase in Coxiella burnetii transformants.
Coxiella burnetii can be transformed to ampicillin resistance by electroporation with plasmids encoding beta-lactamase. However, non-plasmid emergence of resistance to ampicillin also develops. To validate the usefulness of the bla gene marker for selection and detection, transformed C. burnetii were examined for beta-lactamase expression by use of immunoblotting after SDS-PAGE. The 29-kDa mature form of the beta-lactamase protein was detected in C. burnetii lysates. Quantitation of these immunoblot signals showed that C. burnetii surprisingly expressed low levels of beta-lactamase. The results validate the use of plasmid-encoded ampicillin resistance as a means for selecting C. burnetii transformants; they also suggest that levels of ampicillin used for selection pressure should be empirically determined and that detection of beta-lactamase by antibody blotting done to confirm transformants.
Suhan
M L
ML
Department of Microbiology and Immunology, Robert E. Byrd Health Sciences Center, West Virginia University, P.O. box 9177, Morgantown, WV 26506, USA.
Thompson
H A
HA
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D016997:Coxiella burnetii / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D006224:Cricetinae; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D014169:Transformation, Bacterial; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D001618:beta-Lactamases