A high-efficiency recombineering system with pcr-based ssdna in bacillus subtilis mediated by the native phage recombinase gp35
文献类型：期刊论文
| 作者 | Sun, Zhaopeng1,2; Deng, Aihua1; Hu, Ting1; Wu, Jie1,2; Sun, Qinyun1,2; Bai, Hua1,2; Zhang, Guoqiang1; Wen, Tingyi1 |
| 刊名 | Applied microbiology and biotechnology
 |
| 出版日期 | 2015-06-01 |
| 卷号 | 99期号:12页码:5151-5162 |
| 关键词 | Bacillus subtilis Recombinase Recombineering frequency Pcr-based ssdna |
| ISSN号 | 0175-7598 |
| DOI | 10.1007/s00253-015-6485-5 |
| 通讯作者 | Wen, tingyi(wenty@im.ac.cn) |
| 英文摘要 | Bacillus subtilis and its closely related species are important strains for industry, agriculture, and medicine. however, it is difficult to perform genetic manipulations using the endogenous recombination machinery. in many bacteria, phage recombineering systems have been employed to improve recombineering frequencies. to date, an efficient phage recombineering system for b. subtilis has not been reported. here, we, for the first time, identified that gp35 from the native phage spp1 exhibited a high recombination activity in b. subtilis. on this basis, we developed a high-efficiency gp35-meditated recombineering system. taking single-stranded dna (ssdna) as a recombineering substrate, ten recombinases from diverse sources were investigated in b. subtilis w168. gp35 showed the highest recombineering frequency (1.71 +/- 0.15 x 10(-1)). besides targeting the purine nucleoside phosphorylase gene (deod), we also demonstrated the utility of gp35 and beta from escherichia coli lambda phage by deleting the alpha-amylase gene (amye) and uracil phosphoribosyltransferase gene (upp). in all three genetic loci, gp35 exhibited a higher frequency than beta. moreover, a phylogenetic tree comparing the kinship of different recombinase hosts with b. subtilis was constructed, and the relationship between the recombineering frequency and the kinship of the host was further analyzed. the results suggested that closer kinship to b. subtilis resulted in higher frequency in b. subtilis. in conclusion, the recombinase from native phage or prophage can significantly promote the genetic recombineering frequency in its host, providing an effective genetic tool for constructing genetically engineered strains and investigating bacterial physiology. |
| WOS关键词 | GRAM-POSITIVE BACTERIA ; ESCHERICHIA-COLI ; HOMOLOGOUS RECOMBINATION ; CHROMOSOMAL INTEGRATION ; TRANSFORMATION ; ELECTROPORATION ; IDENTIFICATION ; MYCOBACTERIA ; MUTAGENESIS ; COMPETENCE |
| WOS研究方向 | Biotechnology & Applied Microbiology |
| WOS类目 | Biotechnology & Applied Microbiology |
| 语种 | 英语 |
| WOS记录号 | WOS:000355208900017 |
| 出版者 | SPRINGER |
| URI标识 | http://www.irgrid.ac.cn/handle/1471x/2376606 |
| 专题 | 中国科学院大学 |
| 通讯作者 | Wen, Tingyi |
| 作者单位 | 1.Chinese Acad Sci, CAS Key Lab Microbial Physiol & Metab Engn, Inst Microbiol, Beijing 100101, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China |
| 推荐引用方式 GB/T 7714 | Sun, Zhaopeng,Deng, Aihua,Hu, Ting,et al. A high-efficiency recombineering system with pcr-based ssdna in bacillus subtilis mediated by the native phage recombinase gp35[J]. Applied microbiology and biotechnology,2015,99(12):5151-5162. |
| APA | Sun, Zhaopeng.,Deng, Aihua.,Hu, Ting.,Wu, Jie.,Sun, Qinyun.,...&Wen, Tingyi.(2015).A high-efficiency recombineering system with pcr-based ssdna in bacillus subtilis mediated by the native phage recombinase gp35.Applied microbiology and biotechnology,99(12),5151-5162. |
| MLA | Sun, Zhaopeng,et al."A high-efficiency recombineering system with pcr-based ssdna in bacillus subtilis mediated by the native phage recombinase gp35".Applied microbiology and biotechnology 99.12(2015):5151-5162. |
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来源：中国科学院大学
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