Effect Of Integration Site On Gene Expression On The Chromosome Of Escherichia Coli

Escherichia coli is one of the most well known model organisms,and its chromosome structure and genome sequence have been completely parsing.Because of its simple operation and rapid propagation,it has become the most commonly used host bacteria in genetic engineering.The location of a gene on chromosome determines the nature of the gene,and changing the location of the gene may alter the host's basic features or gene expression.In this paper,we study the effect of integration sites on gene expression by integrating reporter genes into different positions on chromosomes.The Red recombinant system of lambda bacteriophage has become an important tool in genetic engineering.As its convenience and high efficiency,it has been widely used for gene integration,replacement and gene knockout in Escherichia coli chromosome.There are also non-essential regions in the genome of Escherichia coli.However,some non-essential regions are not specific function,and the deletion of these sequences has no effect on the stability of the organism and gene expression.Therefore,in this study,we used the Red homologous recombination system to replace the reporter gene lacZ into the non-essential region of the chromosome of E.coli,so as to study the effect of the position of chromosome on gene expression.Ten non essential regions flanked by the DH1 chromosome of Escherichia coli oriC were selected to be the integration site of the reporter gene lacZ.Integration of reporter gene at different sites by two step double strand breaks to promote homologous recombination.In this method,two donor plasmids were constructed and transfected into the cells by the method of transformation to complete the two step homologous recombination process.The first step:Transforming donor plasmid p15AD2IC-XLR and helper plasmid to DH1-qlacZ competent cell,and then L-arabia sugar induced helper plasmid expressing Red recombinase and homing endonuclease?-Cre ?.Homing endonuclease ?-Cre ? cleavage donor plasmid p15AD2IC-XLR releasing Cm resistance gene fragment.Under the action of Red recombinant enzyme,the Cm resistant fragment replaced the non-essential region gene on chromosome,and completed the first step homologous recombination.The second step:Co-transforming donor plasmid pBRIS-CP61acZ and helper plasmid pKAISGBE to positive bacteria of the first step.?-Sce ? homing endonuclease and Red recombinase were producted by L-ara inducing the helper plasmid.?-Sce ? homing endonucleaseacted on the ?-Sce? specific sites of the donor plasmid pBRIS-CP61acZ and then produced the CP61acZ.The red recombinase promoted the CP61acZ replacing the Cm resistance gene fragment,and the reporter gene lacZ was successfully integrated into the designated regions of the Escherichia coli chromosome.Through the identification of PCR,the host bacteria DHl-XCP6lacZ were obtained.Ten different experimental strains were obtained by the two steps of homologous recombination.According to the reaction of beta galactosidase with ONPG,we can detect the expression level of lacZ gene.Real time fluorescent quantitative PCR was used to detect the level of transcription and gene copy number of lacZ gene.All experimental results were shown that the closer the gene position is to the replication initiation site,the higher the level of gene expression,transcription level and copy number,and the result is not affected by gene orientation.In order to investigate whether this conclusion has universal applicability,we study the expression of onHpL gene,which is an anti VEGF antibody Fab fragment expressed by Para-T7 promoter,integrated into the chromosome of different sites.ELISA was used to detect the expression of Fab fragments,and the results were consistent with the expected results confirming the previous conclusion again.