Effect Of Promoter Flanking Sequence On T7 Promoter Function

Objective:To investigate the effect of different flanking sequences at the 5' end of the T7 promoter on the transcriptional activity of the T7 promoter.Methods:1.Using the plasmid PET-42a(+)as a template,the T7 promoter in the plasmid was amplified by overlapping PCR,and different short-chain repeats(ie 4 bp C,5 bp A,5 bp G or 5 bp T)were inserted at the 5'-side close to the T7 promoter.Both ends of the PCR fragment contain XbaI,sphI cleavage sites,respectively.2.The PCR fragment and plasmid PET-42a(+)were digested with restriction endonucleases,and the products were purified with a Purification Kit and an ultrafiltration centrifuge tube,respectively.The purified PCR and plasmid fragments were then ligated with T4 DNA ligase to construct a recombinant plasmid containing the different repeat sequences in the 5' flanking sequence of the T7 promoter.3.The reconstituted plasmid was transformed into a cloned host DH5? competent cell,the plasmid was amplified,and the strain was sequenced and stored.4.The recombinant plasmid was extracted,transformed into competent cells of expression strain BL21(DE3),and the strain was sequenced and preserved.5.BL21(DE3)strain containing the expression plasmid of interest was induced with different concentrations of IPTG,and then bacterial total RNA and protein were extracted using a kit.6.The expression levels of GST-Tag mRNA and protein were detected by qRT-PCR and Western blotting(WB),respectively.7.Results of qRT-PCR were statistically analyzed by using SPSS 24.0.Results: 1.The sequencing results showed that the expression plasmid PET-42a(+)with the correct target sequence at the 5' end of the T7 promoter was successfully constructed.2.qRT-PCR results of GST-Tag mRNA: There was no significant difference in GST-Tag mRNA levels between different groups of BL21(DE3)strains that contained different plasmid vectors after induced by 1 mM IPTG.Compared with the negative control,the GST-Tag mRNA of strain induced by IPTG was significantly increased.3.The protein level of GST-Tag investigated by Western blot:(1)When induced by IPTG 0.11 mM-1 mM for 0.5-1 h,there was no significance of the GST-Tag protein level among different strains.Compared with the negative control,the GST-Tag protein level of the IPTG-induced strain was significantly increased.(2)when induced by IPTG at 0.028-0.05 mM for 20 min,the level of GST-Tag protein in each group ranked as follow : Po>Pa>Pt>Pc>Pg.The GST-Tag protein levels of IPTG-induced strain were significantly higher compared to the negative control.Conclusions:Under normal conditions,the flanking sequence at the 5' side of the T7 promoter has little effect on the transcriptional efficiency of the T7 promoter,but when the concentration of the inducer or T7 RNA polymerase is decreased,the transcriptional efficiency of the T7 promoter can be affected.This finding suggests that changes in the flanking sequence of the promoter may be one of the causes of certain individual differences such as disease susceptibility and the like.