Cloning, Construction Of Expression Vector And Prokaryotic Expression Of S. Meliloti PcoR Gene

In long-term heavy metal contaminated environment, many microbes produce high resistance to a wide range of heavy metals and composites, resulting in induction of many heavy metal resistance genes and proteins. Metal genes can activate and coding metallothionein, operon,metal transport enzymes and permease, heavy metals through the combination of these substances to form crystals or inactivation mechanisms such as the promotion of excretion of heavy metals detoxification of heavy metals.PcoR gene synthesis according to GenBank, Klebsiella pneumoniae (GeneID: NP₉43472) gene, were designed and synthesized one pair of primers (synthesized by the TaKaRa Company) for the amplification PcoR gene. PCR reaction program was: 95℃5min; 94℃1min 30 s; 58℃1min 30 s; 72℃3 min; 34 cycles; final extension at 72℃10min. Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit to prepare for digestion. According to the instructions of DNA gel extraction kit, recover purified PCR product. The recovered PCR products were inserted into pGEM-Teasy vector, the recombinant plasmid pGEM-PcoR, transformed into DH5αcompetent cells, 37℃train 12 16 h. Picked white colonies were inoculated in LB broth containing Amp, 37℃shaking culture 12 16 h, extraction of recombinant plasmid PCR and restriction enzyme digestion. With EcoR I and Hind III digested pET-41a vector and PcoR gene fragment was recovered after digestion, in the role of T4 DNA ligase to do under the direction connection. The products were transformed into BL21 competent cells, coated plates, select monoclonal colony PCR. Colony PCR, enzyme restriction shall contain both positive recombinant clone pET-41a-PcoR positive clones. Choose a clone sent for sequencing Technology Co., Ltd. Nanjing Jin Site. With pET-41a as the control empty plasmid was transformed into bacteria, the DNA sequencing after plasmid into BL21 RIL competent cells, containing 100 mg / L ampicillin (Amp) of the LB culture plate, 37℃incubated overnight. The next day, picked from the LB culture plate containing the recombinant plasmid, empty vector transformed E. coli bacteria and one each were inoculated into 5 mL LB medium (containing 100 mg / L Amp), 37℃shaking culture. When bacteria OD600 value = 0.5, in accordance with IPTG final concentration of 1.0 mmol / L induced at different times (1,2,3,4 and 5h), or different IPTG concentrations (0.1,0.25,0.5,0.75 and 1.0mmol / L ) under the induction of 5h, more recombinant protein production, to determine the optimal induction conditions.BLASTn analysis of copper resistance genes showed the homology of the copper resistance gene on plasmid or the genome of Sinorhizobium pcoR and E. coli, E. sukiyaki, K. pneumonia, and S. marcescens is 99%. Anti-copper protein BLASTp homology search and analysis showed the homology of anti-copper protein pcoR and E. coli, K. pneumonia and transcriptional regulation protein pcoR of K. pneumonia subsp, two-component response regulators in anti-copper of S. marcescens is 100%, homology with Booker's bacteria, Ralston bacteria and Pseudomonas and other heavy metal response regulator is more than 57%.The length of 687 bp of Sinorhizobium pcoR gene can be successfully synthesized by PCR. Recombinant pET-41a-PcoR in E. coli strain BL21 (RIL) in 28℃, 200 r / min by 1mmol / L IPTG induction 5 h, express a large number of soluble fusion protein GST-PcoR.