Rational design of a thermo-inducible near-zero-background T-vector platform based on PhiX174 lysis gene E.

T vectors are core tools for TA cloning and are widely used for the direct cloning of PCR products. In the present study, a novel versatile T vector, designated pZBT01C, was developed based on a modified lysis gene E (mE) from bacteriophage PhiX174, which allows only positive tranformants to grow. Ten unique cloning sites were introduced into the mE gene, flanked by two XcmI sites. Universal M13-48/M13-47 primer binding sites were incorporated 35 bp upstream and downstream of the TA cloning site, and the enhancer T7g10 and Shine-Dalgarno box were placed upstream to stabilize mE expression. With a compact size of 3029 bp, pZBT01C is prepared by XcmI digestion to generate 3'-T overhangs. At the induction temperature, the plasmid pZBT01C exhibited strong host-killing activity. In cloning tests, all randomly selected 20 colonies were confirmed to be positive by colony PCR. The potential of pZBT01C as an expression system was further demonstrated by temperature-inducible expression of the reporter gene GFP. Owing to its near-zero-background performance and versatile design, pZBT01C is expected to have broad application prospects in PCR product cloning and related molecular biology applications.