Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl2 is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0 degrees C42 degrees C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42 degrees C0 degrees C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.

中文翻译：

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质粒DNA如何在大肠杆菌的人工转化过程中渗透细胞膜？

在CaCl2存在下用质粒DNA人工转化大肠杆菌是重组DNA技术中广泛使用的技术。但是，DNA跨细胞膜转移的确切机制在很大程度上不清楚。在这项研究中，结合到细胞外膜上的荧光染料三甲基铵二苯基己三烯（TMA-DPH）的稳态和时间分辨各向异性的测量结果表明，标准转化过程中的热脉冲（0摄氏度至42摄氏度）步骤已经大大降低了细胞的外膜流动性。流动性的下降是由于脂质从细胞表面释放到细胞外介质所致。随后对细胞的冷休克（42摄氏度）将流动性进一步提高到其原始值，这是由于膜蛋白释放到细胞外介质中引起的。当重复热脉冲和冷冲击步骤的循环时，分别发生了更多的脂质和蛋白质释放，最终直至第三循环逐渐提高了转化效率。通过原子力显微镜对感受态细胞表面的研究表明，脂质的释放在细胞表面形成了孔。而且，热脉冲步骤几乎使细胞内膜去极化。在此交流中，我们认为热脉冲步骤对DNA的进入有两个重要作用：（a）脂质的释放和随后在细胞表面上形成的孔的形成，这有助于DNA穿过外膜屏障，以及（b）降低膜电位，这有助于DNA穿过大肠杆菌的内膜。直至第三周期，最终最终提高了转换效率。通过原子力显微镜对感受态细胞表面的研究表明，脂质的释放在细胞表面形成了孔。而且，热脉冲步骤几乎使细胞内膜去极化。在此交流中，我们认为热脉冲步骤对DNA的进入有两个重要作用：（a）脂质的释放和随后在细胞表面上形成的孔的形成，这有助于DNA穿过外膜屏障，以及（b）降低膜电位，这有助于DNA穿过大肠杆菌的内膜。直至第三周期，最终最终提高了转换效率。通过原子力显微镜对感受态细胞表面的研究表明，脂质的释放在细胞表面形成了孔。而且，热脉冲步骤几乎使细胞内膜去极化。在此交流中，我们认为热脉冲步骤对DNA的进入有两个重要作用：（a）脂质的释放和随后在细胞表面上形成的孔的形成，这有助于DNA穿过外膜屏障，以及（b）降低膜电位，这有助于DNA穿过大肠杆菌的内膜。