首页|Controlling expression and inhibiting function of the toxin reporter for simple detection of the promoters' activities in Escherichia coli
Controlling expression and inhibiting function of the toxin reporter for simple detection of the promoters' activities in Escherichia coli
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NSTL
Elsevier
The naturally occurring and mutated promoters inserted into expression plasmids or Escherichia coli chromosome are essential for recombinant protein production and metabolic engineering. Analyzing their activities and screening the promoter libraries require the simple and easy-to-use reporter. Here, we developed a novel and efficient approach to detect the promoter activity, based on E. coli cell growth inhibited by overexpression of bacteriophage phi X174 gene E product (LyE), but recovered by pre-overexpression of Bacillus subtilis MraY (BsMraY). Under the conditional LyE construct expression in the absence or the presence of the BsMraY, activities of promoters including the reported P-T7/lac, P-tac, P-BAD, P-rha, P-hucR, P-prpB, P-cum, the wild type and engineered Ptet for leaky and induced expression, the PthrC for auto-induction, and the P-ms for constitutive expression were assayed. In one-plasmid coexpression system, the P-BAD promoter activity detected using the reporter gene was related to the insertion site. The constructed LyE toxic effects were correlated with toxin expression levels, as determined by the split green fluorescent protein reconstitution. Microscopic analysis showed that cells lysis occurred by the LyE induced with arabinose. Taken together, the toxin reporter construct is a convenient and cost-effective tool to examine the promoter activity in E. coli.
NSTL
Elsevier
