title: A non-restricting and non-methylating Escherichia coli strain for DNA cloning and high-throughput conjugation to Streptomyces coelicolor.
abstract: Escherichia coli strains are used in secondary metabolism research for DNA cloning and transferring plasmids by intergeneric conjugation. Non-restricting strains are desirable for DNA cloning and non-methylating strains are beneficial for transferring DNA to methyl-restricting hosts, like Streptomyces coelicolor. We have constructed a non-methylating E. coli strain, JTU007, by deleting the DNA methylation genes dcm and dam from the widely used non-restricting cloning host DH10B. JTU007 was tested as donor for the conjugative transfer of a plasmid containing the 39 kb actinorhodin biosynthesis gene cluster to S. lividans and S. coelicolor. The Dcm⁻ Dam⁻ strain JTU007 transferred DNA into S. coelicolor A(3)2 derivatives at high frequency. To demonstrate the usefulness of E. coli JTU007 for gene cloning, we constructed a comprehensive S. toxytricini genomic cosmid library, and transferred it using high-throughput conjugation to the methyl-restricting S. coelicolor. One of the cosmid clones produced a brown pigment, and the clone was revealed to carry a tyrosinase operon. JTU007 is more useful than ET12567 because it does not restrict methylated DNA in primary cloning, and gives higher transformation and cosmid infection frequencies.
authors: State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors: Hao
authors: H
authors: Zhou
authors: Yemin
authors: Y
authors: Wang
authors: Yanfei
authors: Y
authors: Yu
authors: Tingli
authors: T
authors: Bai
authors: Li
authors: L
authors: Chen
authors: Pei
authors: P
authors: Liu
authors: Hang
authors: H
authors: Guo
authors: Chenchen
authors: C
authors: Zhu
authors: Meifeng
authors: M
authors: Tao
authors: Zixin
authors: Z
authors: Deng
mesh_terms: D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D019175:DNA Methylation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins