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Gene silencing in E scherichia coli using antisense RNA s expressed from doxycycline‐inducible vectors
Author(s) -
Nakashima N.,
Tamura T.
Publication year - 2013
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12066
Subject(s) - antisense rna , biology , doxycycline , gene , gene silencing , t7 rna polymerase , gene expression , microbiology and biotechnology , sense (electronics) , rna , non coding rna , rna silencing , escherichia coli , rna interference , genetics , bacteriophage , chemistry , antibiotics
Abstract Here, we report on the construction of doxycycline (tetracycline analogue)‐inducible vectors that express antisense RNA s in E scherichia coli . Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNA s from the vectors was more tightly regulated than the previously constructed isopropyl‐β‐D‐galactopyranoside‐inducible vectors. Furthermore, expression levels of antisense RNA s were enhanced by combining the doxycycline‐inducible promoter with the T7 promoter‐T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline‐inducible promoter, was integrated into the lac Z locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications. Significance and Impact of the Study A gene silencing method using antisense RNA s in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNA s, allowing choice of a vector appropriate for the target genes or experimental purpose.