Counterselection method based on conditional silencing of antitoxin genes in Escherichia coli.
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.
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.
Miyuki
M
Tsukuda
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.
Nobutaka
N
Nakashima
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. Electronic address: miyazaki-kentaro@aist.go.jp.
Kentaro
K
Miyazaki
D000992:Antitoxins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics; D010957:Plasmids / Q000032:analysis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics
D000992:Antitoxins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005819:Genetic Markers; C555039:MqsA protein, E coli; C508571:MqsR protein, E coli; D016372:RNA, Antisense; C525943:RnlA protein, E coli; C557568:rnlB protein, E coli
Antisense RNA; Antitoxin; Counterselection; Escherichia coli; MqsA; MqsR/MqsA; RnlA/RnlB; RnlB; Toxin–antitoxin