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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

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.
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f3: 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.

Mentions: To find the reasons of higher efficiency in room temperature protocol, electron microscopy was used for comparative analysis of the morphological shapes of cold competent cells and room temperature competent cells of E. coli. Their comparative analysis showed that cold competent cells appeared to shrink more than room temperature cells, and the surface of room temperature competent cells was found smoother (Fig 3a–d). Shrunken cells might be more difficult to transform, and the bacterial cell membrane and wall could be more permeable for foreign DNA entry at a higher temperature. Additionally, it may be difficult for the shrunken cells to form pores that allow DNA transfer through the cell membrane under electroporation conditions, and after electroporation most of the cold competent cells were found to be lysed. (Fig. 3e,f). From this we assume that the bacterial cell membrane/cell wall might have better permeability for foreign material to enter into the cell.


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)

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

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

f3: 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.
Mentions: To find the reasons of higher efficiency in room temperature protocol, electron microscopy was used for comparative analysis of the morphological shapes of cold competent cells and room temperature competent cells of E. coli. Their comparative analysis showed that cold competent cells appeared to shrink more than room temperature cells, and the surface of room temperature competent cells was found smoother (Fig 3a–d). Shrunken cells might be more difficult to transform, and the bacterial cell membrane and wall could be more permeable for foreign DNA entry at a higher temperature. Additionally, it may be difficult for the shrunken cells to form pores that allow DNA transfer through the cell membrane under electroporation conditions, and after electroporation most of the cold competent cells were found to be lysed. (Fig. 3e,f). From this we assume that the bacterial cell membrane/cell wall might have better permeability for foreign material to enter into the cell.

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