Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency

Abstract

Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples.

Figure 1. Transformation efficiency of competent cells.

(a) Effect of temperature, E. coli GB2005 cells transformed by ~0.1 μg of pGB-amp-Ptet-plu1880 (27.8 kb) were plated on Amp plates. 1, the normal ice-cold method for preparing electrocompetent cells; 2, as for 1 but the cells were kept on ice for 15 min before electroporation; 3, as for 1 but the cells were placed at room temperature (RT) for 15min before electroporation; all cuvettes were used at RT; 4, every step was done at RT; 5, no plasmid DNA. (b) RT prepared cells were transformed with different plasmids. 1, pBR322 origin with ampicillin resistance (27.8 kb); 2, p15A origin with chloramphenicol resistant (54.7 kb); 3, p15A origin with ampicillin resistance (54.7 kb); 4, BAC with chloramphenicol resistant (>120 kb); 5, BAC with kanamycin resistant (91.7 kb); 6, BAC with ampicillin resistant (91.7 kb). (c) Different E. coli strains tested for electroporation transformation. Cells were transformed by 0.1 μg of pGB-amp-Ptet-plu1880 and plated on Amp plates. Error bars, SD; n = 3.

Figure 2. Recombineering using room temperature electrocompetent cells.

(a) Colony number of a standard LLHR assay in GB05-dir from the normal and the cold method in E. coli. (b) As for A, but with pBR322 origin. (c) As for A, but with a standard LCHR assay in GB05-red. (d) The electrocompetent cells were prepared on ice first. After adding PCR product kan cassette into the ice-cold electrocompetent cells, the cells plus DNA mixture were shifted to RT for 3 minutes before electroporation (middle column). CT, Cold temperature; RT, Room temperature. Error bars, SD; n = 3.

Figure 3. Phenotypes of the cells by room temperature and cold protocols and also electroporated the cells for subsequent analysis by electron microscopy.

(a–d): micrograph of the cells washed with cold and room temperature protocols with different magnications. (e–f): micrograph of the electroporated cells mixture with episomal insertion DNA.