McCleary William R
Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
Appl Microbiol Biotechnol. 2009 Sep;84(4):641-8. doi: 10.1007/s00253-009-2137-y. Epub 2009 Jul 25.
The ability to control the expression of chromosomal genes is important for many applications, including metabolic engineering and the functional analysis of cellular processes. This mini-review presents recent work on the application of techniques that allow researchers to replace a chromosomal promoter with one designed for a specific level of activity, thereby exerting precise transcriptional control while retaining the natural genetic context of a gene or operon. This technique, termed promoter swapping, involves the creation of a PCR product that encodes a removable antibiotic resistance cassette and an engineered promoter. Short homology sequences on the ends of the PCR fragment target it for homologous recombination with the chromosome catalyzed by phage-derived recombination proteins. After the PCR product is introduced by electroporation into an appropriate acceptor strain, antibiotic resistance selects the desired recombination products. The antibiotic resistance cassette is then removed from the strain by site-specific recombination leaving the engineered promoter precisely positioned upstream of a target gene but downstream of a short scar consisting of a single site-specific recombination site.
控制染色体基因表达的能力对于许多应用都很重要，包括代谢工程和细胞过程的功能分析。本综述介绍了近期的研究工作，这些研究涉及应用一些技术，使研究人员能够用设计为具有特定活性水平的启动子替换染色体启动子，从而在保留基因或操纵子自然遗传背景的同时实现精确的转录控制。这种技术称为启动子交换，涉及创建一个编码可去除抗生素抗性盒和工程化启动子的PCR产物。PCR片段末端的短同源序列使其能够与由噬菌体衍生的重组蛋白催化的染色体进行同源重组。通过电穿孔将PCR产物引入合适的受体菌株后，抗生素抗性可选择所需的重组产物。然后通过位点特异性重组从菌株中去除抗生素抗性盒，使工程化启动子精确地定位在靶基因的上游，但在由单个位点特异性重组位点组成的短疤痕下游。
