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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

Recombineering using room temperature electrocompetent cells.(a) Colony number of a standard LLHR assay17 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.
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f2: Recombineering using room temperature electrocompetent cells.(a) Colony number of a standard LLHR assay17 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.

Mentions: The plasmid transformation efficiency significantly increased by room temperature electrocompetent cells was not the destination. It was necessary to evaluate the improvement of the room temperature protocol on lambda Red or Rac RecET mediated recombineering. A simple assay using a PCR product of linear vector (p15A ori plus cm or pBR322 ori plus cm) and a PCR product with kanamycin (kan) was built to test LLHR efficiency17. E. coli strain GB05-dir with recET on its chromosome was used for LLHR test25. The results showed that LLHR in room temperature competent cells was 6–10 times more efficient than the cells prepared on ice. Both p15A origin and pBR322 origin plasmids gained the same fold increase (Fig. 2a,b). A direct cloning experiment to fish out the thailandepsin gene cluster (~39 kb) from Burkholderia thailandensis had been performed, around 150 colonies were obtained by using cold electrocompetent cells, but by using room temperature electrocompetent cells more than 600 colonies were obtained. This improvement leads to a higher chance to clone large DNA fragments from genomic DNA pools directly.


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)

Recombineering using room temperature electrocompetent cells.(a) Colony number of a standard LLHR assay17 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Recombineering using room temperature electrocompetent cells.(a) Colony number of a standard LLHR assay17 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.
Mentions: The plasmid transformation efficiency significantly increased by room temperature electrocompetent cells was not the destination. It was necessary to evaluate the improvement of the room temperature protocol on lambda Red or Rac RecET mediated recombineering. A simple assay using a PCR product of linear vector (p15A ori plus cm or pBR322 ori plus cm) and a PCR product with kanamycin (kan) was built to test LLHR efficiency17. E. coli strain GB05-dir with recET on its chromosome was used for LLHR test25. The results showed that LLHR in room temperature competent cells was 6–10 times more efficient than the cells prepared on ice. Both p15A origin and pBR322 origin plasmids gained the same fold increase (Fig. 2a,b). A direct cloning experiment to fish out the thailandepsin gene cluster (~39 kb) from Burkholderia thailandensis had been performed, around 150 colonies were obtained by using cold electrocompetent cells, but by using room temperature electrocompetent cells more than 600 colonies were obtained. This improvement leads to a higher chance to clone large DNA fragments from genomic DNA pools directly.

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