Construction Of Bacteria Expression Vector With Red Fluorescent Reporter Gene

In order to make expression of foreign protein in bacteria more efficient, rapid and convenient, we select red fluorescent protein (RFP) as reporter gene to construct a new bacteria expression vector, hoping to discriminate recombinant and non-recombinant colonies with naked eye on the second day after transformation of ligation:colonies without foreign gene would show red color, while colonies containing foreign gene would not show red color. At last we find a best expression vector, through researching different types of promoter, DNA sequence of RFP, inducer and amino acid sequence of RFP.Firstly, we used T7, lac and tac promoter expression system:after inserting red fluorescent protein gene (DsRed2) into the different vectors, BL21(DE3) host strain was transformed. Result showed that recombinant colonies would turn red after several days. And there is no obvious difference between the three kinds of promoter.By analysis of RFP (DsRed2) gene sequence, we found two adverse factors in the gene:(1) the GC content of the gene is high and unevenly distributed, this distribution would make the transmission of RFP gene information unfavorable;(2) all the proline codons in DsRed2are CCC, which is the least used in bacteria. These two adverse factors would make high level expression of RFP difficult. So we optimized DsRed2gene sequence and synthesized the whole gene, hoping to accelerate the red colony through increasing the expression level of RFP. Results showed that the recombinant colonies containing the new synthesized RFP gene would turn red faster than before.We also tried to use inducers to make red colony appear rapidly. By using different type and concentration of the inducers, results showed that galactose really could accelerate red appearance speed.Finally, through analyzing the amino acid sequence of RFP we found that mutations at several sites favored RFP structure maturation. So we introduced7point(S4T,N6D,V7D,H41T,N42Q,V44A,A145P) mutation into the DsRed2gene (both original and synthesized ones). But all the7point mutation recombinants showed no progress in the colony red-turning speed. After reevaluating the7point mutation, we speculated that A145P mutation might dramatically changed RFP structure, and this would be unfavorable for RFP colony turn red. So we finally choose3point mutation (H41T,N42Q,V44A) and adopted8different recombination designs. Fortunately, with the synthesized DsRed2gene, the3point mutation designs gave positive vectors: on the second day of transformation, colonies containing3point mutated synthesized DsRed2gene turn red. The best vector was named30a17cZMCRFP.