fulltext.study @t Gmail

Counterselection method based on conditional silencing of antitoxin genes in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
20180 43161 2015 5 PDF Available
Title
Counterselection method based on conditional silencing of antitoxin genes in Escherichia coli
Abstract

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 asRNAMqsA as a counterselection marker was demonstrated by efficient plasmid curing and strain improvement experiments.

Keywords
Counterselection; Escherichia coli; Antitoxin; Toxin–antitoxin; Antisense RNA; MqsA; RnlB; MqsR/MqsA; RnlA/RnlB
First Page Preview
Counterselection method based on conditional silencing of antitoxin genes in Escherichia coli
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 120, Issue 5, November 2015, Pages 591–595
Authors
, , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering