外源载体高效转化肺炎克雷伯菌的新途径

Highly efficient transformation with plasmid DNA in Klebsiella pneumoniae

摘要:

研究介绍了提高Klebsiella pneumoniae电转化效率的新途径,即直接从固体平板上收集K.pneumoniae菌落制备电转化感受态细胞,完全不同于传统的试验方法。试验菌株为野生型K.pneumoniae NTUH-K2044和magA—突变型菌株。将大小不同的质粒pIP843T、pIP843TdhaB、pIP843TdhaT电转化K.pneumoniae,计算电转化效率。电转化试验结果表明:K.pneumoniaeNTUH-K2044固体菌电转化效率高达2×10⁵±300转化子/μgDNA,而其液体菌电转化效率仅为150±10转化子/?gDNA;其magA—突变株固体菌的转化效率最高,可以达到3.4×10⁷±500转化子/μgDNA,比液体菌电转化效率提高了104倍。同时发现质粒大小对电转化效率并没有明显影响。此外,激光共聚焦显微镜观察发现固体平板和液体培养基中的菌体存在形态学方面差异,推测固体培养菌电转化效率的显著提高和形态学方面的表现可能具有一定的相关性。

Abstract:

Attempts to transform Klebsiella pneumoniae resulted in very low efficiencies because of capsule polysaccharide (CPS). It was reported that some chelating agents could reduce CPS production and improve transformation efficiency. These methods mentioned above could not improve transformation efficiency apparently by incorporating such agents to liquid medium. However, this method introduces a simple way for efficient transformation of K. pneumoniae. In this method, K. pneumoniae strains NTUH-K2044 and magA- mutant are envolved as recipients. The plasmids used in this way are composed of pIP843T，pIP843TdhaB，pIP843TdhaT with different sizes. The sole critical step is to harvest bacteria on LB plates to prepare competent cells. 150±10, 1.3×10³±100, 2×10⁵±300, and 3.4×10⁷±500 transformants were obtained per microgram plasmid DNA with NTUH-K2044 liquid cells, magA^- liquid cells, NTUH-K2044 solid cells, and magA^- solid cells, respectively. The number of transformants per μg DNA obtained by electroplating solid cells is at least 103 fold higher than that of transformants with liquid-cultured bacteria. This method will benefit gene manipulation and genetic study in K. pneumoniae.