Construction Of T7 RNA Polymerase Gene Expression System In Anabaena 7120 And Expression Of GCS

Cyanobacteria are photosynthetic prokaryotes with many characteristics.Their photosynthetic metabolic structure makes them rather appropriate to be industrialized chassis cells.The cultivation of cyanobacteria has the characteristics of low cost,simple operation,and no toxicity to human body.The biggest limitation of cyanobacteria as a bioengineered basal cell still lies in the relatively low expression of exogenous genes.The T7 RNA polymerase expression system is a highly efficient prokaryotic expression system derived from E.coli T7 phage.Currently,the pET vector derived from the T7 polymerase expression system has been successfully commercialized.Some studies have successfully constructed a variety of eukaryotic expression cell lines with this expression system,but due to the differences in eukaryotic prokaryotic,there is currently no eukaryotic T7 polymerase expression system put into production.OBJECTIVE:To verify the transgenic cyanobacterium containing T7 RNA 1 polymerase,construct a shuttle expression vector expressing GCSF gene,and transform it into cyanobacteria containing T7 RNA polymerase.Through screening and identification,the complete cyanobacteria of T7 RNA polymerase expression system was obtained.METHODS:Molecular biology methods were used to identify the transgenic cyanobacteria,vector construction,and triparental transfer.The cyanobacteria containing the target expression system were obtained,identified and observed for growth status.result:1.PCR,RT-PCR and WB methods were used to verify the cyanobacteria transferred into T7 RNA polymerase.The experimental results showed that the T7 RNA polymerase gene was successfully transferred into Anabaena 7120,resulting in significant differences in cyanobacterial protein expression and wild type.;2.Construction of shuttle expression vector,introduction of T7 promoter by GCSF gene pre-PCR in the laboratory,and restriction endonuclease digestion of pRL-489 vector without promoter,so that only the T7 promoter was constructed in the shuttle expression vector.There are promoters.After constructing the vector and transferring it into HB101 strain,the vector was verified by PCR and enzyme digestion methods.3.The constructed shuttle expression vector was introduced into cyanobacteria using a triparental junction method.Different control groups were set up to investigate the transfer of cyanobacteria with the T7 RNA polymerase gene and transfer into wild-type cyanobacteria,and transfer into the vector with the T7 promoter and growth after using the original vector.4.Screening after transformation to verify gene transfer and GCSF expression.The obtained transformants showed a phenomenon of growth discoloration.After observing the transformant that grew red,it was found that it was a single cell,and the growth rate was compared with that of the wild type pRL-T7-GCSF vector to be confirmed.5.The cyanobacteria positive with PCR result were compared for growth and induced growth.Induction was induced using IPTG at a concentration of 1 mmol.Changes in cyanobacteria growth within one week were examined.It was found that IPTG inhibited the growth of cyanobacteria,and the growth was significantly less than that after adding.Two weeks after the addition of IPTG,the cyanobacterial cells will rupture and the intracellular material will be released.6、After semi-quantitative RT-PCR and Western Bloting,it was found that expression of exogenous proteins was detected after the cyanobacterium containing T7 RNA polymerase was transferred into the GCSF gene containing the T7 promoter.Gray scale analysis was performed and expression efficiency was found to be higher than that of the expression vector using the PpsbA promoter.There is improvement.Conclusion:Validation showed that the T7 RNA polymerase gene was successfully transferred into Anabaena 7120,which can be used as a follow-up experimental material.A pRL-T7-GCSF shuttle expression vector with a T7 promoter was obtained and successfully transferred into a cyanobacterium harboring the T7 RNA polymerase gene.