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Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

Tu Q, Yin J, Fu J, Herrmann J, Li Y, Yin Y, Stewart AF, Müller R, Zhang Y - Sci Rep (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f1: 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.

Mentions: It was inconvenient to maintain low temperature conditions for preparation, storage and transport of the electrocompetent cells. We intended to test the transformation efficiency of the electrocompetent cells prepared at room temperature. A large plasmid pGB-amp-Ptet-plu1880 (27.8 kb) was transformed into E. coli GB2005 strain1724 at various temperature. The warm electrocompetent cells showed 10 times higher transformation efficiency than the cold electrocompetent cells (Fig. 1a). After placing the cold electrocompetent cells at room temperature for 15 minutes, the transformation efficiency increased by 5 folds (Fig. S1a). In contrast, after the room temperature electrocompetent cells were placed on ice for 15 minutes before electroporation, there was a significant decrease in transformation efficiency (Fig. S1b).


Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

Tu Q, Yin J, Fu J, Herrmann J, Li Y, Yin Y, Stewart AF, Müller R, Zhang Y - Sci Rep (2016)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837392&req=5

f1: 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.
Mentions: It was inconvenient to maintain low temperature conditions for preparation, storage and transport of the electrocompetent cells. We intended to test the transformation efficiency of the electrocompetent cells prepared at room temperature. A large plasmid pGB-amp-Ptet-plu1880 (27.8 kb) was transformed into E. coli GB2005 strain1724 at various temperature. The warm electrocompetent cells showed 10 times higher transformation efficiency than the cold electrocompetent cells (Fig. 1a). After placing the cold electrocompetent cells at room temperature for 15 minutes, the transformation efficiency increased by 5 folds (Fig. S1a). In contrast, after the room temperature electrocompetent cells were placed on ice for 15 minutes before electroporation, there was a significant decrease in transformation efficiency (Fig. S1b).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.

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.

No MeSH data available.


Related in: MedlinePlus