Karunakaran P, Endresen D T, Ertesvåg H, Blatny J M, Valla S
UNIGEN Center for Molecular Biology and Department of Biotechnology, Norwegian University of Science and Technology, 7489, Trondheim, Norway.
FEMS Microbiol Lett. 1999 Nov 15;180(2):221-7. doi: 10.1111/j.1574-6968.1999.tb08799.x.
TrfA is the only plasmid-encoded protein required for RK2 replication. We report here the construction and characterization of an RK2-based vector in which trfA is expressed from the inducible promoter Pm. The resulting construct, pJBSD1, was found to replicate in Escherichia coli DH5a (recA(-)) only in the presence of a Pm inducer. In two tested E. coli recA(+) strains pJBSD1 could replicate in the absence of inducer, but a replication inducer-dependent phenotype was obtained in these strains by introducing a mutation known to reduce the trfA expression level. The plasmid construct could be used as a conditional suicide vector system for targeted chromosomal integration via homologous recombination. This feature may potentially be used for many types of studies in microbial molecular biology.
TrfA是RK2复制所需的唯一质粒编码蛋白。我们在此报告一种基于RK2的载体的构建和特性，其中trfA由可诱导启动子Pm表达。所得构建体pJBSD1仅在Pm诱导剂存在的情况下才能在大肠杆菌DH5a（recA(-)）中复制。在两个测试的大肠杆菌recA(+)菌株中，pJBSD1在没有诱导剂的情况下也能复制，但通过引入已知会降低trfA表达水平的突变，在这些菌株中获得了依赖复制诱导剂的表型。该质粒构建体可作为一种条件性自杀载体系统，用于通过同源重组进行靶向染色体整合。这一特性可能潜在地用于微生物分子生物学的多种研究类型。
