Study On The Factors Affecting The Preparation And Transformation Of E.coli BL21(DE3)competent Cells

In recent years,genetic engineering has been applied to modern agriculture,industry,food fermentation engineering,environmental protection and biological medicine on a large scale,which has greatly changed the lifestyle of people.Cloning and expression of exogenous genes is one of the most commonly used techniques in genetic engineering research.The preparation and transformation of competent cells from E.coli has become a basic experimental method frequently used in the molecular biology experiments.The preparation of high efficient and stable competent E.coli cells is an important guarantee for the subsequent gene cloning and gene expression.The transformation efficiency of competent E.coli cells plays a very important role in the success or failure of subsequent experiments.E.coli BL21(DE3)is often used as host bacteria expressing exogenous genes.It is of practical value to study the preparation and transformation of E.coli BL21(DE3)competent cells.The preparation and transformation of competent E.coli is a complex process of biophysical and chemical reactions.Different genotypes of bacteria,the growth state of the bacteria,the preparation methods and conditions of competent cells,the experimental parameters of the transformation process,the preservation technology,the size of the plasmid and other experimental factors can affect the transformation results.In this paper,the factors affecting the preparation and transformation of competent E.coli BL21(DE3)were systematically studied,and the TSS method was optimized to master the key experimental parameters that need to be controlled in the preparation and transformation experiments of E.coli BL21(DE3).It can be concluded from this study that:(1)Glycerobacteria of E.coli BL21(DE3)preserved for two years were cultured overnight in LB liquid medium,and then transferred and activated twice.The growth state after culture at 30?for 2-3 hours was the best period for preparation of competent state;(2)The optimal transformation period of E.coli BL21(DE3)occurs when OD₆₀₀=0.4-0.5,that is,in the middle and early stage of logarithmic growth.At this time,the number of live E.coli BL21(DE3)could reach 1×10⁸ cells/m L;When OD₆₀₀=0.46,the conversion efficiency of p UC19 plasmid was 4.6×10⁶CFU/?g by CaCl₂ method;(3)E.coli BL21(DE3)with OD₆₀₀=0.46 was treated with TSS buffer,and competent cells were prepared by 5×KCM(a mixture of KCl,CaCl2 and Mg Cl2)was added into the transformation system,and LB medium containing 0.2 mol/L glucose was added in the resuscitation process.Under certain transformation conditions,the plasmid p UC19 could be transformed into 1.48×10⁷ cfu/?g plasmid DNA;(4)The optimal experimental conditions for preparing receptive states and plasmid transformation using the optimized TSS method:E.coli BL21(DE3)broth cultured to OD600=0.46 was frozen in a sterile precooled centrifuge tube for 30min,centrifuged at 4200r/min for 10min at 4?,then the thilli were collected,TSS buffer of 1/10 of the original bacterial broth was added,the cells were suspended and packed to 100 ul,and stored at-70?.Plasmid 5?L,5×KCM 20?L,deionized water 75?L were added into the cultured cells,and the cells were gently mixed.The cells were frozen for 30 min and placed at room temperature for 10 min.The cells were added with 400?L LB medium containing 0.2 mol/L glucose,and the cells were shaken at37?,180 r/min.Recovery for 40 min;The transformed product was coated with 200?L and cultured overnight for 12-16 h;(5)The conversion efficiency of p UC19 plasmid was 3.2 times higher than that of CaCl₂ method when the receptive state was prepared by optimized TSS method.