摘要
基因敲除技术是了解基因功能的重要技术手段。以大肠杆菌K-12 MG1655基因组hfq(309 bp)和rne-710(1056 bp)基因为模型,首先构建Δhfq∷Spe和Δrne-710∷Spe菌株,通过融合PCR方法分别构建缺失hfq(309 bp)和rne-710(1056 bp)的融合片段并连接至辅助质粒,缺失hfq和rne-710的片段经重组分别替换壮观霉素抗性盒,得到无痕敲除株Δhfq和Δrne-710。双质粒无痕敲除和融合PCR方法相结合为大片段基因缺失开辟了新的途径。
Gene modification is an important technique to understand gene function. We firstly constructed Δhfq：：Spe and Δrne-710：：Spe mutant strains of Escherichia coli MG1655. The fragment lacking of hfq and rne-710 was ligated to the auxiliary plasmid and separately replace the spectinomycin box by homologous recombinase system to obtain the Δhfq and Δrne-710 mutant strains. The combination of two-plasmid scarless genetic modification and fusion PCR led to a new way for the long DNA fragment gene deletions.
作者
王净
吕瑞辰
韩延平
杨瑞馥
Jing Wang;Ruichen Lv;Yanping Han;Ruifu Yang(College of Animal Science and Technology, Hebei North University, Zhang/iakou 075131, Hebei, China;State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2018年第4期602-612,共11页
Chinese Journal of Biotechnology
基金
国家重点基础研究发展计划(973计划)(No.2014CB744405)
河北省科技计划项目(No.16236605D-1(2017))资助~~
