Research Of Electroporation Processing Of Lactobacilli Delbrueckii Subsp. Bulgaricus And Lactococcus Lactis With The Vector PMG36e

Lactic acid bacteria widely distributed in nature.Lactic acid fermented foods is the development trend in today's world of food,meet the requirements of the times,has great vitality and broad prospects.The current study of lactic acid bacteria have entered the era of molecular biology and genetic engineering,through genetic engineering,the foreign gene into lactic acid bacteria , given the beneficial properties of lactic acid bacteria.However,lactic acid bacteria are Gram-positive bacteria,with very thick and dense rigid cell wall,resulting in conversion of exogenous DNA into the recipient cell's efficiency is very low,which severely restricts the molecular biology of lactic acid bacteria and genetic engineering technology.Therefore,improving the efficiency of genetic transformation of lactic acid bacteria is lactic acid in molecular biology and genetic engineering in the development of key technical problems be solved.In this paper,we chose the constitutive expression plasmid pMG36e of the lactic acid bacteria as a vector , and the Lactobacilli delbrueckii subsp.bulgaricus Lb-MH and the Lactococcus lactis ML23 as a host,use the method of electroporation to research the different factors on the impact of electroporation efficiency.Factors include wall treatment,plasmid concentration,electroporation parameters,composition of the recovery medium,recovery culture time and the restriction & modification of host.We proved the optimal electroporation conditions,increased transformation efficiency.Results showed that the optimal values of Lb-MH for concentration of glycine and of sucrose and the pulse strength,risistance are 2.5%(W/V) ,0.5 mol/L and 12 kV/cm,200Ω,respectively.Best electroporation efficiency is attained after incubation in 20mmol/L MgCl₂,CaCl₂ containing SMRS broth for another 2 h,combined with the sub-inhibitory antibiotic concentration in the selective plates.When we use the receptor-modified strains of plasmid to electroporation,the transformation efficiency has increased by 30 times,reached 6.3×104 cfu/μg DNA.And the optimal values of ML23 for concentration of glycine and of sucrose and the pulse strength,risistance are 2.0%(W/V) ,0.5mol/L and 11 kV/cm,200Ω,respectively.Best electroporation efficiency is attained after incubation in SMRS broth for another 2 h,combined with the sub-inhibitory antibiotic concentration in the selective plates.When we use the receptor-modified strains of plasmid to electroporation,the transformation efficiency has increased by 15 times,reached 1.05×105 cfu/μg DNA.The research of this paper provided foundation for exogenous plasmid be transformedinto Lactococcus lactis ML23 and Lactobacillus bulgaricus Lb-MH,and also provided foundation for further study of establish high-efficiency expression system of lactic acid bacteria and the development of genetic engineering technology.