Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies
Escherichia coli recA− strains are usually used for cloning to prevent insert instability via RecA-dependent recombination. Here, we report that E. coli BW25113 (recA+) competent cells prepared by using a previously reported transformation and storage solution (TSS) had 100-fold or higher transforma...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-03-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.838698/full |
| _version_ | 1831813081039110144 |
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| author | Yuqing Yang Yuqing Yang Qiaoli Yu Min Wang Rui Zhao Huaiwei Liu Luying Xun Luying Xun Yongzhen Xia |
| author_facet | Yuqing Yang Yuqing Yang Qiaoli Yu Min Wang Rui Zhao Huaiwei Liu Luying Xun Luying Xun Yongzhen Xia |
| author_sort | Yuqing Yang |
| collection | DOAJ |
| description | Escherichia coli recA− strains are usually used for cloning to prevent insert instability via RecA-dependent recombination. Here, we report that E. coli BW25113 (recA+) competent cells prepared by using a previously reported transformation and storage solution (TSS) had 100-fold or higher transformation efficiency than the commonly used E. coli cloning strains, including XL1-Blue MRF’. The cloning success rates with E. coli BW25113 were 440 to 1,267-fold higher than those with E. coli XL1-Blue MRF’ when several inserts were assembled into four vectors by using a simple DNA assembly method. The difference was in part due to RecA, as the recA deletion in E. coli BW25113 reduced the transformation efficiency by 16 folds and cloning success rate by about 10 folds. However, the transformation efficiency and the cloning success rate of the recA deletion mutant of E. coli BW25113 are still 12- and >48-fold higher than those of E. coli XL1-Blue MRF’, which is a commonly used cloning strain. The cloned inserts with different lengths of homologous sequences were assembled into four vectors and transformed into E. coli BW25113, and they were stably maintained in BW25113. Thus, we recommend using E. coli BW25113 for efficient cloning and DNA assembly. |
| first_indexed | 2024-12-22T21:48:32Z |
| format | Article |
| id | doaj.art-dafa76d7748749a89faa0cef64c8b916 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-22T21:48:32Z |
| publishDate | 2022-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-dafa76d7748749a89faa0cef64c8b9162022-12-21T18:11:26ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-03-011310.3389/fmicb.2022.838698838698Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning EfficienciesYuqing Yang0Yuqing Yang1Qiaoli Yu2Min Wang3Rui Zhao4Huaiwei Liu5Luying Xun6Luying Xun7Yongzhen Xia8State Key Laboratory of Microbial Technology, Shandong University, Qingdao, ChinaInstitute of Marine Science and Technology, Shandong University, Qingdao, ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, ChinaSchool of Molecular Biosciences, Washington State University, Pullman, WA, United StatesState Key Laboratory of Microbial Technology, Shandong University, Qingdao, ChinaEscherichia coli recA− strains are usually used for cloning to prevent insert instability via RecA-dependent recombination. Here, we report that E. coli BW25113 (recA+) competent cells prepared by using a previously reported transformation and storage solution (TSS) had 100-fold or higher transformation efficiency than the commonly used E. coli cloning strains, including XL1-Blue MRF’. The cloning success rates with E. coli BW25113 were 440 to 1,267-fold higher than those with E. coli XL1-Blue MRF’ when several inserts were assembled into four vectors by using a simple DNA assembly method. The difference was in part due to RecA, as the recA deletion in E. coli BW25113 reduced the transformation efficiency by 16 folds and cloning success rate by about 10 folds. However, the transformation efficiency and the cloning success rate of the recA deletion mutant of E. coli BW25113 are still 12- and >48-fold higher than those of E. coli XL1-Blue MRF’, which is a commonly used cloning strain. The cloned inserts with different lengths of homologous sequences were assembled into four vectors and transformed into E. coli BW25113, and they were stably maintained in BW25113. Thus, we recommend using E. coli BW25113 for efficient cloning and DNA assembly.https://www.frontiersin.org/articles/10.3389/fmicb.2022.838698/fulltransformationcompetent cellsEscherichia colirecAcloningrecombination |
| spellingShingle | Yuqing Yang Yuqing Yang Qiaoli Yu Min Wang Rui Zhao Huaiwei Liu Luying Xun Luying Xun Yongzhen Xia Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies Frontiers in Microbiology transformation competent cells Escherichia coli recA cloning recombination |
| title | Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies |
| title_full | Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies |
| title_fullStr | Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies |
| title_full_unstemmed | Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies |
| title_short | Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies |
| title_sort | escherichia coli bw25113 competent cells prepared using a simple chemical method have unmatched transformation and cloning efficiencies |
| topic | transformation competent cells Escherichia coli recA cloning recombination |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.838698/full |
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