Highly efficient transformation with plasmid DNA in Klebsiella pneumoniae

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.