Goude Renan, Parish Tanya
Institute of Cell and Molecular Science, Barts and the London, Queen Mary's School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London E1 2AT, UK.
Methods Mol Biol. 2009;465:203-15. doi: 10.1007/978-1-59745-207-6_13.
High-efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall but is compounded by the fact that most molecular techniques have been developed for distantly related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified, and DNA transformation of many mycobacterial species has now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the conditions for the electroporation pulse. Optimized methods for the transformation of both slow-grower and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.
高效转化是分枝杆菌研究中的一个主要限制因素。分枝杆菌属可能难以转化，这主要是由其厚而含蜡质的细胞壁导致的，但大多数分子技术是针对诸如大肠杆菌和枯草芽孢杆菌等远缘物种开发的，这一情况使问题更加复杂。尽管存在这些障碍，但已鉴定出分枝杆菌质粒，并且现在已经描述了许多分枝杆菌物种的DNA转化。将DNA导入分枝杆菌最成功的方法是电穿孔。许多参数有助于成功转化，这些参数包括物种/菌株、转化DNA的性质、所用的选择标记、生长培养基以及电穿孔脉冲的条件。本文详细介绍了慢生长菌和快生长菌转化的优化方法。报告了不同分枝杆菌物种和各种选择标记的转化效率。
