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	A high-efficiency recombineering system with pcr-based ssdna in bacillus subtilis mediated by the native phage recombinase gp35
	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
	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
出版者	SPRINGER
WOS记录号	WOS:000355208900017
内容类型	期刊论文
URI标识	http://www.corc.org.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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