Study On The Establishment Of A E.coli Phage-Defense System Based On CRISPR-Cas9

Phage is a type of virus that can infect bacteria,archaea,algae and other microorganisms.It exists widely in nature and has long been accompanied by the growth and evolution of microorganisms.Synthetic biology technology has greatly improved the ability of microbial cell factories to produce fuels,bulk chemicals and natural products through the creation and optimization of synthetic pathways or the adaptation of chassis cells,thereby replacing petrochemical refining and plant resources extraction.However,at present,when microbial fermentation is carried out in some laboratories or factories,slow growth of microorganisms and reduced yield caused by phage infection may cause losses.Aiming at the scientific question of how to improve the ability of microbial cell factories to defend against phage infection,this study used E.coli as the host,designed and constructed gene circuits based on CRISPR-Cas9.Gene circuits target different sites of the phage genome and cause the degradation of phage DNA through targeted cutting,achieving the role of eliminating the phage.The main research contents are as follows:(1)This study designed and constructed a chronic MKR phage.Through resistance gene screening and red fluorescence screening,the elimination process of phage DNA can be traced simply and efficiently.At the same time,the host-specific restriction is used to reduce the risk of escape infection in the process of phage infection research.In addition,a lytic MKC phage was also designed and constructed.(2)In order to study the clearance effect of gene circuits on the intracellular phage genome in E.coli,this project designed and constructed two different CRISPR-Cas9 gene circuits p GM1 and p GM2,respectively targeting one and two sites on MKR phage DNA.They were introduced into E.coli infected by MKR phage to targeted cutting phage DNA.The MKR phage infection rate in 12 h was calculated.It was confirmed that both p GM1 and p GM2 can effectively remove phage DNA in98% E.coli and p GM2 clears faster.(3)In order to study the effect of gene circuits on the prevention of phage infection of E.coli in the environment,this project preliminarily introduced p GM1 and p GM2 into E.coli that had never been infected by MKR phage,and then added MKR phage to the E.coli culture environment for infection.By studying the phage infection rate in 12 h,it was confirmed that p GM2 can effectively enable E.coli to obtain the ability to prevent phage infection in the environment.This conclusion has also been further verified in the prevention experiment of MKC phage.The results of this study prove that the dual target gene circuit can effectively prevent and eliminate phage infection,and it is expected to be widely used in the design and construction of microbial cell factories in the future to improve the safety and stability of microbial cell factories.