Construction The Rcombinant Escherichia Coli Containing Glucose-6-phosphate Dehydrogenase And Cysteine Synthase For Co(?) Bioaccumulation

Glucose-6-phosphate dehydrogenase?g6pd?is the speed limiting enzyme of pentose phosphate pathway,and Cysteine synthase?cys?participates in the synthesis of important precursors cysteine in the resistance metabolism of organisms,and the synergistic effect of these two genes can maintain the reduced state of the organism and enhance the resistance metabolism.In this paper,g6pd gene and cys gene were recombinant into Escherichia coli by genetic engineering techniques,and the ability to improve the tolerance and adsorption of heavy metals by recombinant strain was studied.This paper studied about the amplification and analysized of g6pd gene and cys gene,constructing a dual expression vector containing glucose-6-phosphate dehydrogenase and cysteine synthetase,obtaining recombinant it into Escherichia coli,and screening recombinant Escherichia coli by inducible expression.The adsorption capacity of cobalt ions and its resistance capacity were also investigated.These results are as following:1.PCR primers were designed based on the sequences of g6pd gene and cys gene on the NCBI database,and two genes fragment were amplified.After sequencing,the homology analysis of the sequence with DNAman showed that two genes of amplified sequences were respectively the g6pd gene and cys gene were amplified from Rhodopseudomonas palustris.Analysis and simulation of the amino acid sequence and protein conformation revealed that the protein expressed by the g6pd contains 448 amino acids with 50.18 kDa weight,and the cys contains 332 amino acids with 34.5 kDa weight.And the three-dimensional structure of the two proteins was simulated by SWISS-MODEL.2.PCR primers of the g6pd gene?containing BamH I and Hind III site?and the cys gene?containing Nde I and Kpn I site?were designed.Recombinant binary expression vector pETD-GC containing a g6pd gene and cys gene was constructed by restriction enzyme digestion and ligation.The constructed plasmids were transferred into Escherichia coli?E.coli?BL21?DE3?and recombinant E.coli contain pETD-GC was screened.3.Screened the recombinant strain BL-GC which can expression recombinant protein correctly though IPTG induction,and investigated its absorption and tolerance of Co²⁺under different pH,temperature and initial concentration of Co²⁺.Results show that the suitable absorption condition of BL-GC was pH=6.5,30?,and the initial concentration of Co²⁺was 60 mg/L.Cultured for 15 hours at this condition,the adsorption rate could reach 39.7%,and the adsorption ability of BL-GC was increased3 folds.When the concentration of Co²⁺reached 120 mg/L,BL-GC could continue to grow,and adsorption rate reached 31.6%.Observed the growth curves of two strains,BL21?DE3?and BL-GC,the experiment was found that the plasmid transferred resulted in a lower biomass of BL-GC than BL21?DE3?.But the adsorption capacity of BL-GC is higher than the host strains,this paper illustrated that recombinant BL-GC,which containing Rhodopseudomonas palustris Glucose-6-phosphate dehydrogenase gene and cysteine synthetase gene,enhanced the host strain's resistance capacity when induced expression successfully.