RRC ID 61165
Author Kuroki A, Ohtani N, Tsuge K, Tomita M, Itaya M.
Title Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector.
Journal Gene
Abstract The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges.
Volume 399(1)
Pages 72-80
Published 2007-9-1
DOI 10.1016/j.gene.2007.04.030
PII S0378-1119(07)00247-8
PMID 17560740
MeSH Bacillus subtilis / genetics* Cloning, Molecular / methods* Conjugation, Genetic Gene Transfer Techniques* Genetic Vectors / genetics* Genome, Bacterial / genetics Plasmids / chemistry Plasmids / genetics* Synechocystis / genetics
IF 2.638
Resource
Prokaryotes B. subtilis MBS794 MBS795 MBS796 MBS797 MBS798 MBS799 MBS800 MBS801 MBS802 MBS803 MBS804 MBS805 MBS806