Williams S G, Cranenburgh R M, Weiss A M, Wrighton C J, Sherratt D J, Hanak J A
Cobra Therapeutics Limited, The Science Park, University of Keele, Keele, Staffordshire ST5 5SP, UK.
Nucleic Acids Res. 1998 May 1;26(9):2120-4. doi: 10.1093/nar/26.9.2120.
The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.
重组质粒在细菌宿主中，尤其是在大肠杆菌中的增殖，对于克隆DNA的扩增与操作以及重组蛋白的生产至关重要。传统上，细菌转化体的分离及随后的质粒稳定维持是通过质粒携带的选择标记基因来完成的。在此，我们描述了一种新型系统，该系统利用质粒介导的阻遏物滴定来激活染色体选择标记，从而不再需要质粒携带的标记基因。构建了一种经过修饰的大肠杆菌宿主菌株，其包含一个在乳糖操纵子/启动子（lac O/P）控制下的条件必需染色体基因（kan）。在没有诱导剂（别乳糖或异丙基-β-D-硫代半乳糖苷）的情况下，该菌株DH1 lackan由于LacI蛋白与lac O/P结合抑制了kan的表达，无法在含卡那霉素的培养基上生长。用含有乳糖操纵子（lac O）的高拷贝数质粒进行转化，通过从操纵子滴定LacI，有效地诱导了kan的表达。因此，该菌株允许选择不含抗生素抗性基因的质粒（它们只需要含有lac O和一个复制起点），这对于用作基因治疗载体具有明显优势。以同样的方式调控一个必需的内源性细菌基因，将能够生产不含残留抗生素污染的重组治疗药物。
