乳酸乳球菌电击转化方法的优化  

Optimization of Electrotransformation of Lactococcus lactis

作　　者：刘亚欣 王宇[1] 杨诺 郭桂英 李迁[3] 曾纪锋[4] 郑继平[1] LIU Yaxin;WANG Yu;YANG Nuo;GUO Guiying;LI Qian;ZENG Jifeng;ZHENG Jiping(Institute of Life Sciences and Pharmacy,Hainan University,Haikou,Hainan 570228;Academic Affairs Office,Hainan University,Haikou,Hainan 570228;Network and Technology Center,Hainan University,Haikou,Hainan 570228;Institute of Animal Science and Technology,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]海南大学生命科学与药学院,海口570228 [2]海南大学教务处,海口570228 [3]海南大学网络与技术中心,海口570228 [4]海南大学动物科技学院,海口570228

出　　处：《热带生物学报》2020年第2期245-250,共6页Journal of Tropical Biology

基　　金：国家自然科学基金(31460699);海南自然科学基金创新研究团队项目(2017CXTD005);海南自然科学基金面上项目(317071);海南大学研究生教学经费(J100029019106004001)。

摘　　要：乳酸菌NICE系统是食品级表达系统,本实验以该系统的表达载体pNZ8148和宿主菌NZ9000为研究对象,对电击转化方法进行了优化。结果表明,在培养基中加入2.5%甘氨酸,收集OD600值为0.3的细胞、用配方Ⅰ[(洗涤液Ⅰ:蔗糖0.5 mol·L-1+甘油10%;洗涤液Ⅱ:蔗糖0.5 mol·L-1+甘油10%+EDTA50 mmol·L-1)]洗涤细胞,再转入0.2μg质粒,利用2.5 kV电压和0.2 cm电击杯进行电击转化,恢复培养1.5 h后转化效率最高,达1.6×108 CFU·μg-1。本实验为乳酸乳球菌NZ9000的电击转化参数提供了数据参考,为基于乳酸菌NICE系统的深入研究奠定了基础。The nisin-controlled gene expression system(NICE)is a food-grade system used for gene expression in Lactococcus lactis.Electrotransformation was optimized via the NICE,consisting of an expression vector pNZ8148 and a host strain Lactococcus lactis NZ9000.The results showed that the electrotransformation of L.lactis NZ9000 with the expression vector pHZ8148 gave the highest efficiency(1.6×10^8 CFUμg−1)when L.lactis NZ9000 was cultured on the medium added with 2.5%of glycine,and its competent cells grown to 0.3 OD600 were prepared by washing with the liquid detergent formulation I(Sucrose 0.5 mol·L^−1+glycerol 10%),transferred into with 0.2μg plasmid of pNA8148,electrotranformed at 2.5 kV on a 0.2 cm gap cuvette,and mixed gently for 1.5 h before plating.This study provides optimum conditions for electrotransformation of L.lactis NZ9000 which are favorable for further study of L.lactis based on the NICE.

关 键 词：乳酸乳球菌 电击转化 电击转化优化 

分 类 号：S129[农业科学—农业基础科学]