Mather M W, McReynolds L M, Yu C A
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078, USA.
Gene. 1995 Apr 14;156(1):85-8. doi: 10.1016/0378-1119(95)00074-g.
A mobilizable, broad-host-range (bhr) plasmid was derived from the widely used IncP1 vector pRK415. The new vector, pRKD418, contains an additional resistance gene and an enlarged multiple cloning site (MCS) region. The optimal growth of pRK415-containing bacteria under photosynthetic conditions generally requires the use of optical filters to protect the selective antibiotic tetracycline (Tc) from photooxidation with the resulting production of toxic photoproducts; pRK415 is not stably maintained in the absence of selective pressure. The addition of a trimethoprim-resistant dihydrofolate reductase-encoding gene provided for optimal photosynthetic growth in the presence of a selective antibiotic without any special apparatus. The presence of an antibiotic marker not found in commonly used cloning vectors in many cases facilitates the subcloning of inserts into the bhr plasmid. The new MCS region provides further cloning flexibility with at least sixteen available restriction sites. Easily constructed derivative plasmids, exemplified by pRKD418KmE, provide a convenient screening procedure for the detection of recombinants during subcloning.
一种可移动的广宿主范围（bhr）质粒源自广泛使用的IncP1载体pRK415。新载体pRKD418含有一个额外的抗性基因和一个扩大的多克隆位点（MCS）区域。在光合条件下，含pRK415的细菌的最佳生长通常需要使用滤光片来保护选择性抗生素四环素（Tc）免受光氧化，以避免产生有毒的光产物；在没有选择压力的情况下，pRK415不能稳定维持。添加一个编码耐甲氧苄啶的二氢叶酸还原酶基因，可在有选择性抗生素存在的情况下实现最佳光合生长，而无需任何特殊设备。在许多情况下，常用克隆载体中未发现的抗生素标记的存在有助于将插入片段亚克隆到bhr质粒中。新的MCS区域提供了更大的克隆灵活性，至少有16个可用的限制酶切位点。以pRKD418KmE为例的易于构建的衍生质粒，为亚克隆过程中重组体的检测提供了方便的筛选程序。
