Norgard M V, Keem K, Monahan J J
Gene. 1978 Jul;3(4):279-92. doi: 10.1016/0378-1119(78)90038-0.
The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA.
对大肠杆菌菌株chi1776被pBR322质粒DNA转化的敏感性进行了检测和优化。当细胞从L肉汤中收获时浓度为5.0 - 6.0×10⁷ cfu/ml，然后在冷的0.1M NaCl + 5mM MgCl₂ + 5mM Tris（pH 7.6）中洗涤两次，可实现对四环素（Tc）抗性的最大转化。在D - 环丝氨酸（Cyc）而非萘啶酸（Nx）存在下生长的细胞转化效果明显更好。洗涤液和氯化钙溶液中5mM Mg²⁺离子的存在使转化提高约2倍。转化的最佳条件包括pH范围为7.25 - 7.75以及细胞与DNA的比例约为1.6×10⁸ cfu/ng质粒DNA。当细胞在与DNA混合之前，先在250mM KCl + 5mM MgCl₂ + 5mM Tris（pH 7.6）中暴露于100mM CaCl₂时，转化频率最高。细胞 + DNA混合物在冰上孵育60分钟可产生最大数量的Tcr转化体。在我们的实验中，37℃或42℃的热休克处理不同时间都会使转化降至最佳水平的约一半。此外，当细胞 + DNA混合物铺在预冷（4℃）的Tc琼脂平板上时，转化体的回收率最佳。当每平板仅铺5微升细胞 + DNA混合物时，铺板效率最佳，这表明选择性培养基上的非存活背景chi1776细胞会抑制转化体的回收。还发现细胞 + DNA混合物中线性DNA分子的存在会显著抑制pBR322质粒DNA对chi1776的转化。基于这些发现，提出了一种通过pBR322质粒DNA对大肠杆菌chi1776进行质粒特异性转化的新方法。使用该技术使我们能够获得超过10⁷转化体/微克pBR322质粒DNA的转化频率。
