TY - JOUR
T1 - Optimization of electroporation conditions for Mycobacterium avium
AU - Lee, S. H.
AU - Cheung, M.
AU - Irani, V.
AU - Carroll, J. D.
AU - Inamine, J. M.
AU - Howe, W. R.
AU - Maslow, Joel N.
PY - 2002
Y1 - 2002
N2 - Successful transformation and subsequent genetic manipulation of Mycobacterium avium requires suitable vectors, efficient transformation systems, and reliable selectable markers. A systematic analysis of the parameters involved in the transformation of M. avium was performed to optimize DNA transfer. Factors examined included the composition of the growth medium, growth medium additives, variations in washing of the bacteria prior to electroporation, and conditions of electroporation. Of the parameters assayed, the frequency of transformation (defined as the number of transformants per 106 transformed bacteria) showed the greatest increase with the addition of 1.5% glycine to the M. avium culture medium and the use of higher concentrations of plasmid DNA. The addition of 0.5 M sucrose to the growth medium and wash solution yielded a modest increase in transformation frequency, but more importantly afforded greater consistency of results between different batches of cells with no decrease in transformation yields following freezing and thawing. We also confirmed that gfp could be used as a selective marker for M. avium, even as a single copy integrant, and allowed for rapid discrimination between false and true transformants. Using this protocol, we were able to transform nine of 11 clinical strains of M. avium.
AB - Successful transformation and subsequent genetic manipulation of Mycobacterium avium requires suitable vectors, efficient transformation systems, and reliable selectable markers. A systematic analysis of the parameters involved in the transformation of M. avium was performed to optimize DNA transfer. Factors examined included the composition of the growth medium, growth medium additives, variations in washing of the bacteria prior to electroporation, and conditions of electroporation. Of the parameters assayed, the frequency of transformation (defined as the number of transformants per 106 transformed bacteria) showed the greatest increase with the addition of 1.5% glycine to the M. avium culture medium and the use of higher concentrations of plasmid DNA. The addition of 0.5 M sucrose to the growth medium and wash solution yielded a modest increase in transformation frequency, but more importantly afforded greater consistency of results between different batches of cells with no decrease in transformation yields following freezing and thawing. We also confirmed that gfp could be used as a selective marker for M. avium, even as a single copy integrant, and allowed for rapid discrimination between false and true transformants. Using this protocol, we were able to transform nine of 11 clinical strains of M. avium.
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U2 - 10.1054/tube.2002.0335
DO - 10.1054/tube.2002.0335
M3 - Article
C2 - 12464488
AN - SCOPUS:0036452156
SN - 1472-9792
VL - 82
SP - 167
EP - 174
JO - Tuberculosis
JF - Tuberculosis
IS - 4-5
ER -