| Lactic acid bacteria are widely existed in nature and have vital relationship withmankind. Apart from the minority of pathogenic bacteria, most of the lactic acid bacteriahave the function of enhancing the physical ability of human, plus with essentialapplication value among the area of food and medicine industry. Thus, using lactic acidbacteria to product food has broad prospects and has been an inevitable trend of the foodindustry development in the whole world as well. Currently, with the rapid developmentof biology technology and genetic engineering currently, it is not meet the requirementsof researching the traditional physiological functions of lactic acid bacteria but to makethe genetic modification then attach them new biological activity. Besides, the studyabout making lactic acid bacteria as receptor strain of recombinant vector has beenachieved successfully to some extent nowadays, which leads researchers to add lacticacid bacteria with exogenous genes and to show expression of the exogenous genesthrough DNA recombination technology, making such lactic acid bacteria contains newexcellent performance. Nevertheless, lactic acid bacteria is known to be gram-positivebacteria, which has thick as well as rigid cell wall, making it a very low efficiency whentransfer exogenous DNA into the recipient cells, which severely restricts the molecularbiology and genetic engineering technology of lactic acid bacteria. So that, it is a key toimprove the efficiency of genetic transformation, which chould solve lactic acidbacteria’s development in molecular biology as well as genetic engineering. In this paper,we use pMG36e of the vector, at the same time use Lactobacillus delbrueckii subsp.bulgaricus MH, Lactobacillus rhamnosus05-28and Lactobacillus casei05-20as hostsby using electroporation method to research the various factors which impact theelectroporation efficiency. The factors are as follow: proper cell age of the receptorstrains, wall treatment, solutions, the concentration of lysozyme, the concentration ofplasmid, electroporation factors and recovery medium． We proved the optimalelectroporation situations as well as the high transformation efficiency additionally.Additionally, we estabalished a universal method which is suitable for transformingLactobacillus as Lactobacillus delbrueckii subsp. bulgaricus MH, Lactobacillusrhamnosus05-28and Lactobacillus casei05-20.The results are as follow: firstly, for the optimal values of Lactobacillusdelbrueckii.bulgaricus MH, cultivating it under MRS when it grows to middlelogarithmic growth period (8hours), then switching it to the MRS containing glycineand sucrose (concentrations are2%and0.5M respectively), making it growscontinuously until arrive middle logarithmic growth period again (8hours). Harvestingcells and wash them by using the solution of SMM then making enzymolysis of cells with lysozyme (30mg/mL) made by SMM under37℃until it happens to beapproximately60%of protoplasts through microscope. Adding1μL plasmids pMG36e,with the concentration of1μg/μL, into the protoplasts and the electroporation parametersare7.5kV/cm,200,25μF. Best electroporation efficiency is as follow: using properincubation in20mmol/L MgCl2, CaCl2and sucrose of0.5M containing MRS broth(without Tween80) for at least2hours, adding sub-inhibitory antibiotic into theselective plates. As a result, the transformation efficiency reaches1.43×105cfu/μgDNA.Secondly, for the optimal values of Lactobacillus rhamnosus05-28, cultivating it underMRS when it grows to middle logarithmic growth period (9hours), then switching it tothe MRS containing glycine and sucrose (concentrations are2%and0.5Mrespectively), making it grows continuously until arrive middle logarithmic growthperiod again (8hours). Harvesting cells and wash them by using the solution of SMMthen making enzymolysis of cells with lysozyme (30mg/mL) made by SMM under37℃until it happens to be approximately60%of protoplasts through microscope. Adding1μL plasmids pMG36e, with the concentration of1μg/μL, into the protoplasts and theelectroporation parameters are8kV/cm,200,25μF. Best electroporation efficiency isas follow: using proper incubation in20mmol/L MgCl2, CaCl2and sucrose of0.5Mcontaining MRS broth(without Tween80) for at least2hours, adding sub-inhibitoryantibiotic into the selective plates. As a result, the transformation efficiency reaches1.66×105cfu/μgDNA. Thirdly, for the optimal values of Lactobacillus casei05-20,cultivating it under MRS when it grows to middle logarithmic growth period (4hours),then switching it to the MRS containing glycine and sucrose (concentrations are0.5%and0.5M respectively), making it grows continuously until arrive middle logarithmicgrowth period again (4hours). Harvesting cells and wash them by using the solution ofSMM then making enzymolysis of cells with lysozyme (20mg/mL) made by SMMunder37℃until it happens to be approximately60%of protoplasts throughmicroscope. Adding1μL plasmids pMG36e, with the concentration of1μg/μL, into theprotoplasts and the electroporation parameters are9kV/cm,200,25μF Bestelectroporation efficiency is as follow: using proper incubation in20mmol/L MgCl2,CaCl2and sucrose of0.5M containing MRS broth(without Tween80) for at least2hours, adding sub-inhibitory antibiotic into the selective plates. As a result, thetransformation efficiency goes to1.28×105cfu/μgDNA.The study of this thesis provides us the basis for transforming exogenous plasmidinto the Lactobacillus delbrueckii subsp. bulgaricus MH, the Lactobacillus rhamnosus05-28and the Lactobacillus casei05-20, also provides the genetic breeding and thestrain development of lactic acid bacteria, plus with the basic for further research onmaking high-level expression system by using lactic acid bacteria. |
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