Screening Of Anti-bacterialgenes And Anti-phage By Using Yeast And Escherichia Coli Expression System

Pichia yeast and Escherichia coli respectively is eukaryotic and prokaryotic.These two species and their mutant strains are the key eukaryotic and prokaryotic expression systems.Pichia pastoris expression system can express some foreign complex proteins efficiently.In this study,we successfully constructed a cDNA Library of Pinellia ternata.A total of 1080 monoclonal transformants were obtained.In our experiments,two aspects of the screening were performed on the 1080 transformants,which are:1)confrontation test between Rhizoctonia solani and the transformants to screen resistance genes;2)screening of autolysis genes from these transformants.In this study,we performed PCR tests for the library,and proved that it is a high quality library.Screening of genes by using the method of drawing lines is an effective method.In this study,a strain of E.coli phage was isolated from waste water After purification,the morphology was observed by electron microscope,the shape demonstrated that it belongs to a long tailed phage.Sequence analysis proved that it is a Escherichia phage JMPW1.E.coli expression system is the most commonly used expression system.BL21(DE3)possesses the T7RNA polymerase gene,and it is a lysogenic bacterium of ?phage.PET expression system has a T7 promoter,and expression of foreign genes in downstream of the T7 promoter was highly induced by IPTG In this study,the pET expression system(pET-22b(+))was applied,and BL21(DE3)was the host strain.The cDNA Library of Pinellia ternata was successfully constructed.A total of 1380 monoclonal transformants were obtained.By the same PCR method,we proved that it is also a high quality library.In this study,a number of antiphage genes were screened in the constructed cDNA library of E.coli.Three screening methods were explored and tested:1)treatment with mitomycin;2)plasmid transformation method;and 3)shake out again and then drawing lines.