Study On The Expression Level Of Genes Of Bicistron Expression Vector In E.coli

In the post-genome era, more and more functional genes of clear Biological significance will be cloned and applied to engineered organisms for genetic improvement. However,the vast majority traits and physiological functions of organisms will be achieved by the coordination expression of multi-functional genes. Therefore, the coordination expression strategy of multiple genes will become the mainstream of gene engineering on theory study and practical application. The construction of polycistron expression vector and research on exogenous gene expression efficiency effected by different spacer of polycistron in prokaryotes will be important to study on multi-gene expression. Whereas, the quantitative analysis of foreign gene expression level effected by prokaryotes spacers has not been reported.This study intends to analyse the spacer sequences of polycistron of genome in E. coli,and to choose spacer sequences TAAATG and TAATG to study . A series of expression vectors with a bicistron of green fluorescent protein (GFP) and trx were constructed in E. coli, including bicistron expression vector pET32a-Trx-GFPD (spacer sequence is TAAATG), pET32a-Trx–GFPL(spacer sequence is TAATG) and pET32a-Trx-GFPR(no spacer). Control plasmid pET32a-GFP and pET32a-Trx are also constructed. These constructions were transformed to BL21, and IPTG were used to induced to gene expression. The result sugessted that average fluorescence intensive ratio of pET32a-GFP, pET32a-Trx-GFPR, pET32a-Trx-GFPL, pET32a-Trx-GFPD and pET32a-Trx is 970.35, 762.82, 12.974 , 11.810 and 4.4168 respectively. It indicated that different spacer sequence of bicistron have effect on expression level of reporter gene,and it also provide a basis for precise regulation and control of gene expression.