Veselý M, Pátek M, Nesvera J, Cejková A, Masák J, Jirků V
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 14220 Prague 4, Czech Republic.
Appl Microbiol Biotechnol. 2003 Jun;61(5-6):523-7. doi: 10.1007/s00253-003-1230-x. Epub 2003 Feb 20.
The strain Rhodococcus erythropolis CCM2595, which was shown to degrade phenol, was chosen for genetic studies. To facilitate strain improvement using the methods of gene manipulation, the technique of genetic transfer was introduced and cloning vectors were constructed. Using the plasmid pFAJ2574, an electrotransformation procedure yielding up to 7x10(4) transformants/microg DNA was optimized. Escherichia coli- R. erythropolis shuttle vectors were constructed using the replicons pSR1 and pGA1 from Corynebacterium glutamicum. The small vector pSRK21 (5.8 kb) provides six unique cloning sites and selection of recombinant clones using alpha-complementation of beta-galactosidase in E. coli. This vector, exhibiting high segregational stability under non-selective conditions in R. erythropolis CCM2595, was applied to cloning and efficient expression of the gene coding for green fluorescent protein (gfpuv).
已证明能降解苯酚的红平红球菌CCM2595菌株被选用于遗传学研究。为便于使用基因操作方法改良菌株，引入了基因转移技术并构建了克隆载体。利用质粒pFAJ2574，优化了一种电转化程序，每微克DNA可产生多达7×10⁴个转化体。使用谷氨酸棒杆菌的复制子pSR1和pGA1构建了大肠杆菌 - 红平红球菌穿梭载体。小载体pSRK21（5.8 kb）提供六个独特的克隆位点，并利用大肠杆菌中β-半乳糖苷酶的α-互补作用筛选重组克隆。该载体在红平红球菌CCM2595的非选择性条件下表现出高分离稳定性，被用于绿色荧光蛋白（gfpuv）编码基因的克隆和高效表达。
