Tsukuda Miyuki, Nakashima Nobutaka, Miyazaki Kentaro
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Sapporo, Hokkaido 062-8517, Japan; Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, M6-5, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan.
J Biosci Bioeng. 2015 Nov;120(5):591-5. doi: 10.1016/j.jbiosc.2015.03.008. Epub 2015 Apr 23.
Counterselection is a genetic engineering technique to eliminate specific genetic fragments containing selectable marker genes. Although the technique is widely used in bacterial genome engineering and plasmid curing experiments, the repertoire of the markers usable in Escherichia coli is limited. Here we developed a novel counterselection method in E. coli based on antisense RNA (asRNA) technology directed against toxin-antitoxin (TA) modules. Under normal conditions, excess antitoxin neutralizes its cognate toxin and thus the module is stably maintained in the genome. We hypothesised that repression of an antitoxin gene would perturb cell growth due to the toxin being released. We designed asRNAs corresponding to all 19 type II antitoxins encoded in the E. coli genome. asRNAs were then conditionally expressed; repression of MqsA in the MqsR/MqsA module had the greatest inhibitory effect, followed by RnlB in the RnlA/RnlB module. The utility of asRNA(MqsA) as a counterselection marker was demonstrated by efficient plasmid curing and strain improvement experiments.
反选择是一种用于消除含有选择标记基因的特定基因片段的基因工程技术。尽管该技术在细菌基因组工程和质粒消除实验中被广泛应用，但在大肠杆菌中可用的标记物种类有限。在此，我们基于针对毒素-抗毒素（TA）模块的反义RNA（asRNA）技术，开发了一种在大肠杆菌中的新型反选择方法。在正常条件下，过量的抗毒素会中和其同源毒素，因此该模块在基因组中得以稳定维持。我们推测，抗毒素基因的抑制会因毒素释放而干扰细胞生长。我们设计了与大肠杆菌基因组中编码的所有19种II型抗毒素相对应的asRNA。然后条件性表达asRNA；MqsR/MqsA模块中MqsA的抑制具有最大的抑制作用，其次是RnlA/RnlB模块中的RnlB。通过高效的质粒消除和菌株改良实验证明了asRNA（MqsA）作为反选择标记的实用性。
