Construction and Analyses of High Efficient and Broad Host Range Vectors of Pseudomonas fluorescens

Abstract

Pseudomonas fluorescens is a useful common bacterium and has GC content as high as above 60%.Because the genes with high GC content are not easy to be expressed in conventional expression systems,constructing a novel expression system is important in P.fluorescens.The plasmid pCIBhis is commonly used to express protein in P.fluorescens.Since it is above 20 kb in length conjugation is usually used to transfer this plasmid vector into P.fluorescens which results in a more complicated operation.In this study,the replication-related segment and genetic marker gene were amplified from pCIBhis by using PCR and then recombined together with the method called sequence and ligation independent cloning(SLIC) to construct the vector pCIBS1.The transformation method of P.fluorescens was optimized and the plasmid pCIBS1 was electroporated into P.fluorescens.Then T7 and tac promoters were inserted into pCIBS1,respectively,and the expression vectors pCIBS3 and pCIBS2 were constructed.It was found that pCIBS1 could be stable either in P.fluorescens or in Escherichia coli.The green fluorescent protein gene was finally cloned into the two expression vectors and the transformants harboring the recombinant plasmid could express the functional protein in E.coli BL21(DE3).The results lay a foundation for the expression of those genes with high GC content in P.fluorescens.