Department of Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Appl Microbiol Biotechnol. 2010 Jul;87(4):1533-41. doi: 10.1007/s00253-010-2674-4. Epub 2010 May 28.
A method for construction of bacterial strains with multiple DNA inserted into their chromosomes has been developed based on the mini-Mu transposon and FLP/FRT recombination. Exogenous DNA can be integrated by Mu transposition with an FRT cassette containing selection marker and conditional replicative origin (R6Kgammaori). Subsequently, with the introduction of a helper plasmid bearing gene of FLP recombinase, drug-resistant selection marker is excised from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of transposition and excision of selection marker. Each cycle can add further foreign gene(s) to the chromosome. As an example, resistance genes of chloramphenicol, tetracycline, and gentamicin were successively integrated into the chromosome of Escherichia coli BW25113 by three cycles of insertion and excision as described above. This method proved to be simple and time-saving, which could be applicable to a variety of microorganisms.
基于 mini-Mu 转座子和 FLP/FRT 重组，开发了一种将多个 DNA 插入细菌染色体的菌株构建方法。外源 DNA 可以通过含有选择标记和条件复制起点 (R6Kgammaori) 的 FRT 盒的 Mu 转座进行整合。随后，通过引入带有 FLP 重组酶基因的辅助质粒，可从染色体上切除耐药性选择标记。去除辅助质粒的细胞可以进行下一轮转座和选择标记的切除。每个循环都可以将进一步的外源基因添加到染色体中。例如，通过上述插入和切除三个循环，成功地将氯霉素、四环素和庆大霉素的抗性基因相继整合到大肠杆菌 BW25113 的染色体中。该方法简单、省时，适用于多种微生物。
