Construction Of The Regulate Self-Cleaving Escherichia Coli Strain

At present,the commonly used cell wall-breaking techniques include mechanical breaking,ultrasonic method,pyrolysis,chemical method and biological method.Compared with these methods,biological method has the advantages of low energy consumption and mild reaction.E.coli is an important genetically engineered bacteria to express exogenous proteins.It has been found that some biological proteins can cause E.coli cell rupture.Lysozymes in these proteins can induce cell rupture by acting on N-acetylglucosamine and N-acetyl parietal acid.Phage lysin can hydrolyze peptidoglycan in bacterial cell wall to cause cell rupture.Animal pyroptosis-related protein N-terminal fragments can also cleave cells by acting on lipopolysaccharide components on the outer membrane of bacterial cells.This paper is by fusing these three different proteins together to cause E.coli autolysis.By constructing pUC-57Y,pACYC-184YG intermediate vector,successfully synthesized promoter sequence,encoding bacteriophage sp.isolated 181 sequence,RBS ribosome binding site,gasdermin D-N sequence and terminator together,constitute the recombinant fusion gene fragment.Then,it was successfully targeted into E.coli BL-21 genome.The growth rate and lysis efficiency of the constructed recombinant E.coli BL-21 containing recombinant gene fragments,the original E.coli BL-21 and the BL-21 strain containing lysis protein from a pharmaceutical company were determined,and finally the self-regulated lysis E.coli strain was obtained.The results are as follows:(1)The growth performance of E.coli obtained in this study is better than the BL-21 strain containing lysed protein provided by a pharmaceutical company,and the growth rate is about 10 % faster;(2)The constructed strain was induced by the IPTG solution to induce its lysis.The lysis efficiency measured was about 86.77 %.Compared with the lysis rate of a pharmaceutical company's lysis strain,the lysis rate was 76.36 %.The effect is good.In summary,this study successfully constructed a controllable autolysis E.coli strain.The strain itself does not contain plasmids,which can facilitate the expression of exogenous proteins.Autolysis of cells can avoid the inactivation of products when E.coli is broken,save the material and financial resources needed to break the wall,and provide a better host for the production of drug proteins.