Chen Fuwang, Jiang Jie, OuYang Hongsheng, Ma Teng, Peng Zhiyuan, Ma Yunzhi, Chen Xinrong, Pang Daxing, Lin Songyi, Ren Linzhu
Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, 5333 Xi'an Road, Changchun, 130062, People's Republic of China.
Appl Biochem Biotechnol. 2015 Jul;176(5):1472-81. doi: 10.1007/s12010-015-1658-3. Epub 2015 May 9.
Red homologous recombination has been extensively used in recombineering. Because foreign sequences, such as antibiotic resistance genes, FRT-sites, or loxP-sites, are often unwanted in mutant Escherichia coli, we established a markerless deletion system containing short homologous sequences, a positive-selectable marker (kan), and a negative-selectable marker (sacB) for E. coli. For markerless deletion of a specific region of the E. coli genome, a two-step recombination procedure using two different PCR fragments, which were amplified from pUC57-kan-sacB and pUC57-298, was performed. The generation of a pheA-tyrA deficient mutant demonstrated that this markerless deletion system was a simple and efficient method to generate markerless chromosomal deletions in E. coli.
红色同源重组已广泛应用于重组工程。由于在突变型大肠杆菌中，诸如抗生素抗性基因、FRT位点或loxP位点等外源序列通常是不需要的，因此我们建立了一种无标记缺失系统，该系统包含短同源序列、一个正向选择标记（卡那霉素抗性基因）和一个负向选择标记（蔗糖致死基因）用于大肠杆菌。为了对大肠杆菌基因组的特定区域进行无标记缺失，采用了两步重组程序，使用从pUC57-kan-sacB和pUC57-298扩增得到的两个不同的PCR片段。pheA-tyrA缺陷型突变体的产生表明，这种无标记缺失系统是在大肠杆菌中产生无标记染色体缺失的一种简单而有效的方法。
