CAJ | 학술논문

以乳酸菌诱导型表达质粒pNZ8149为载体，Lactococcus lactisNZ3900为受体，采用电转化方法研究电场强度、电击杯规格、受体菌株的生长状态、感受态细胞浓度及分装体积、质粒浓度、电击脉冲时间以及电击杯使用次数等因素对电转化效率的影响。结果表明：在固定电阻200Q、电容25μF条件下，收获对数生长前期的乳球菌制作感受态细胞，并以80μL体积分装，采用2mm规格的电击杯，加入浓度为1．2μg／μL的质粒，在电场强度和脉）中时间分别为10kV／cm和5ms的条件下完成电击转化。转化后的菌液恢复培养2h后涂板计数，转化效率最高可以达到2．6×10^4CFU／μgDNA。研究结果为进一步构建乳酸菌高效表达系统和食品级基因工程乳酸菌株奠定了方法基础。
이유산균유도형표체질립pNZ8149위재체，Lactococcus lactisNZ3900위수체，채용전전화방법연구전장강도、전격배규격、수체균주적생장상태、감수태세포농도급분장체적、질립농도、전격맥충시간이급전격배사용차수등인소대전전화효솔적영향。결과표명：재고정전조200Q、전용25μF조건하，수획대수생장전기적유구균제작감수태세포，병이80μL체적분장，채용2mm규격적전격배，가입농도위1．2μg／μL적질립，재전장강도화맥）중시간분별위10kV／cm화5ms적조건하완성전격전화。전화후적균액회복배양2h후도판계수，전화효솔최고가이체도2．6×10^4CFU／μgDNA。연구결과위진일보구건유산균고효표체계통화식품급기인공정유산균주전정료방법기출。
Using the inducible expression plasmid pNZ8149 of the lactic acid bacteria as a vector, and Lactococcus lactis NZ3900 as a host, the different factors on the impact of electroporation efficiency were studied by the method of electroporation. Factors include electric field strength, cuvette model, cell growth phase, competent cells concentration, volume of competent cells, plasmid concentration, pulse constant and usage counter of cuvette. Results showed that when several parameters in improved protocol were resistance 200 Ω, capacitance 25 μF, the optimum factors were the cell harvested at the early exponential growth phase, 80 μL competent cells, 2 mm cuvette, 1.2 μg/μL plasmid DNA, electrotransformation at the 10 kV/cm and pulse constant for 5 ms. After incubation in GM17MC broth for another 2 h, the transformation efficiency went up to 2.6 × 10^4 CFU/μg DNA with those optimum factors above. The present research provided fundamental method for further study of establish high - efficiency expression system of lactic acid bacteria and the development of genetic engineering technology.