Lee S-H, Cheung M, Irani V, Carroll J D, Inamine J M, Howe W R, Maslow J N
Department of Microbiology, Boston University School of Medicine, Boston, MA, USA.
Tuberculosis (Edinb). 2002;82(4-5):167-74. doi: 10.1054/tube.2002.0335.
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 10(6) 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.
鸟分枝杆菌的成功转化及后续基因操作需要合适的载体、高效的转化系统和可靠的选择标记。为优化DNA转移，对鸟分枝杆菌转化过程中涉及的参数进行了系统分析。所考察的因素包括生长培养基的组成、生长培养基添加剂、电穿孔前细菌洗涤的差异以及电穿孔条件。在所测定的参数中，转化频率（定义为每10⁶个转化细菌中的转化子数）在向鸟分枝杆菌培养基中添加1.5%甘氨酸并使用更高浓度质粒DNA时增加最为显著。向生长培养基和洗涤溶液中添加0.5 M蔗糖使转化频率适度增加，但更重要的是，不同批次细胞之间的结果一致性更高，冻融后转化产量没有下降。我们还证实，绿色荧光蛋白（gfp）可作为鸟分枝杆菌的选择标记，即使作为单拷贝整合体，也能快速区分假转化子和真转化子。使用该方案，我们成功转化了11株鸟分枝杆菌临床菌株中的9株。
