IMPROVED ELECTROPORATION AND CLONING VECTOR SYSTEM FOR GRAM-POSITIVE BACTERIA
IMPROVED ELECTROPORATION AND CLONING VECTOR SYSTEM FOR GRAM-POSITIVE BACTERIA
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DOI：
10.1128/aem.57.4.1194-1201.1991
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发表时间：
1991-04-01
影响因子：
4.4
通讯作者：
LEBLANC, DJ
中科院分区：
文献类型：
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作者：
DUNNY, GM;LEE, LN;LEBLANC, DJ
通过系统地检查各种参数的变化的影响，开发了通过电穿孔转化完整粪肠球菌细胞的方案，所述参数包括（i）生长条件;（ii）电穿孔溶液的组成;（iii）电穿孔条件，例如场强和电阻;（iv）用于转化的DNA的大小、浓度和纯度;（iii）电穿孔溶液的组成;（iv）电穿孔溶液的组成;（iv）电穿孔溶液的组成;（iv）电穿孔溶液的组成;（iv）电穿孔溶液的组成。和（v）用于选择转化体的条件。 该协议的主要特点包括使用指数期细胞生长在抑制浓度的甘氨酸和使用酸性蔗糖电穿孔解决方案。 对于大肠杆菌，每μ g质粒DNA获得> 2 × 10（5）个转化体的频率。粪菌细胞，而链球菌和炭疽杆菌的各种菌株以每μ g质粒DNA 10（3）至10（4）个转化体的频率用相同的方案转化。 构建了一个新的大肠杆菌-链球菌和肠球菌穿梭克隆载体pDL 276，用于与电穿孔系统结合使用。 该载体具有多克隆位点区，侧翼为E。大肠杆菌转录终止序列，一个相对小的大小（< 7 kb），和卡那霉素抗性决定簇中表达的革兰氏阳性和革兰氏阴性宿主。 在大肠杆菌中克隆了各种肠球菌和链球菌的DNA序列。大肠杆菌（包括不能在其它载体上克隆的序列），并通过电穿孔返回到原始宿主。 利用该载体和电穿孔系统直接克隆到E.粪便。
A protocol for transformation of intact Enterococcus faecalis cells by electroporation was developed through a systematic examination of the effects of changes in various parameters, including (i) growth conditions; (ii) composition of the electroporation solution; (iii) electroporation conditions, such as field strength and resistance; (iv) size, concentration, and purity of DNA used for transformation; and (v) conditions used to select for transformants. Key features of this protocol include the use of exponential-phase cells grown in inhibitory concentration of glycine and the use of an acidic sucrose electroporation solution. Frequencies of > 2 x 10(5) transformants per mu-g of plasmid DNA were obtained for E. faecalis cells, whereas various strains of streptococci and Bacillus anthracis were transformed at frequencies of 10(3) to 10(4) transformants per mu-g of plasmid DNA with the same protocol. A novel Escherichia coli-Streptococcus and Enterococcus shuttle cloning vector, pDL276, was constructed for use in conjunction with the electroporation system. This vector features a multiple cloning site region flanked by E. coli transcription termination sequences, a relatively small size (< 7 kb), and a kanamycin resistance determinant expressed in both gram-positive and gram-negative hosts. Various enterococcal and streptococcal DNA sequences were cloned in E. coli (including sequences that could not be cloned on other vectors) and were returned to the original host by electroporation. The vector and electroporation system was also used to clone directly into E. faecalis.