Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter

摘要

Construction of the expression vector pSix1 for strains. The plasmid pYK596, which possesses a hygromycin resistance gene, was digested using RI and I. Two PCR-amplified fragments, a multi-cloning site derived from pET21a, and a putative silica-inducible protein ( ) promoter region [ ] were cloned into pYK596 using an In-Fusion cloning kit. The I site on the pYK596 backbone was deleted by inverse PCR, and the resultant plasmid was named pSix1. To complete the Miller assay, a thermostable β-galactosidase gene from the sp. A4 was cloned downstream of the promoter of pSix1 to yield βgal/pSix1. Sequence of pSix1. The − 35 and − 10 regions of the promoter are underlined. The experimentally determined transcription initiation site (+1) is indicated in bold, and the putative ribosome binding site (rbs) is underlined. Restriction sites in the multi-cloning site and 6× histidine tag sequences are indicated above the sequence