Rohweder Bettina, Semmelmann Florian, Endres Christiane, Sterner Reinhard
Institute of Biophysics and Physical Biochemistry, University of Regensburg, Regensburg, Germany.
Biotechniques. 2018 Jan 1;64(1):24-26. doi: 10.2144/000114628.
Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E. coli strains. Coupling restriction and ligation with the BsaI restriction enzyme minimizes hands-on time, while the need for only three different primers to clone a target gene into the six different vectors keeps overall cloning costs low.
在此,我们对常用表达载体的多克隆位点进行了改造,创建了一套新的克隆质粒,以简化和加速大肠杆菌中的克隆程序。我们的每个标准化质粒都包含两个BsaI限制性酶切位点,可实现高效的基因克隆,并使其表达受T7(pET21a_BsaI、pET28a_BsaI和pMAL-c5T_BsaI)或T5启动子(pUR22和pUR23)的控制。我们这套质粒中的另一个质粒(pTNA_BsaI)可用于生成包含>108个变体的大型基因文库,这些文库可在利用营养缺陷型大肠杆菌菌株的代谢互补进行的选择实验中组成型表达。将限制性酶切和连接与BsaI限制性酶相结合可最大限度地减少实际操作时间,而将目标基因克隆到六种不同载体中仅需三种不同引物,从而使总体克隆成本保持较低水平。