Wang J, Tseng Y H
Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi. 1985 May;18(2):115-22.
We have demonstrated in this study that CaCl2-MnCl2 treatment is effective in the preparation of competent cells of Erwinia chrysanthemi for transformation. Plasmids pMB9 and pBR322, which are of Escherichia coli origins, were transformed into E. chrysanthemi at frequencies of 1.5 X 10(-7) and 4.5 X 10(-7) per recipient, respectively. The frequencies were 60- to 80-fold lower than those in the well established rec- E. coli; however, the procedure is practically useful in transformation experiments. The plasmids were maintained rather stable in the cells if antibiotics were included in the media. When transformed by pECl and pEC6, which were chimeric plasmids consisting of pBR322 and an E. chrysanthemi chromosomal DNA fragment of 2.4 and 3.5 Mdal, respectively, the transformation frequencies of E. chrysanthemi were at the same range of that by pBR322. Neither colony morphology nor the polypectate degradation ability was changed after transformation by the plasmids. In conclusion, we have established a system of molecular cloning in E. chrysanthemi SR120A exploiting pBR322 as a vector.
我们在本研究中证明，氯化钙-氯化锰处理对于制备用于转化的菊欧文氏菌感受态细胞是有效的。源自大肠杆菌的质粒pMB9和pBR322分别以每个受体1.5×10⁻⁷和4.5×10⁻⁷的频率转化到菊欧文氏菌中。这些频率比在成熟的rec⁻大肠杆菌中的频率低60至80倍；然而，该方法在转化实验中实际是有用的。如果培养基中含有抗生素，质粒在细胞中保持相当稳定。当用pECl和pEC6转化时，pECl和pEC6分别是由pBR322和2.4 Mdal及3.5 Mdal的菊欧文氏菌染色体DNA片段组成的嵌合质粒，菊欧文氏菌的转化频率与用pBR322转化时处于相同范围。用这些质粒转化后，菌落形态和多聚半乳糖醛酸降解能力均未改变。总之，我们利用pBR322作为载体建立了菊欧文氏菌SR120A中的分子克隆系统。
