Gittins John R
Ocean and Earth Science, University of Southampton, Waterfront Campus, National Oceanography Centre, Southampton SO14 3ZH, UK.
FEBS Lett. 2015 Jul 8;589(15):1872-8. doi: 10.1016/j.febslet.2015.05.014. Epub 2015 May 14.
A copper resistance gene cluster (6 genes, ∼8.2 kb) was isolated from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery (RR). Following integration of a narrow-host-range plasmid vector adjacent to the target region in the Synechocystis genome (pSYSX), DNA was isolated from transformed cells and the plasmid plus flanking sequence circularized by recombineering to precisely clone the gene cluster. Complementation of a copper-sensitive Escherichia coli mutant demonstrated the functionality of the pcopM gene encoding a copper-binding protein. RR provides a novel alternative method for cloning large DNA fragments from species that can be transformed by homologous recombination.
通过重组工程回收（RR）从蓝藻集胞藻PCC 6803中分离出一个铜抗性基因簇（6个基因，约8.2 kb）。在集胞藻基因组的目标区域附近整合一个窄宿主范围的质粒载体（pSYSX）后，从转化细胞中分离DNA，通过重组工程使质粒加侧翼序列环化，以精确克隆该基因簇。对铜敏感的大肠杆菌突变体的互补实验证明了编码铜结合蛋白的pcopM基因的功能。RR为从可通过同源重组进行转化的物种中克隆大DNA片段提供了一种新的替代方法。
