How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli?
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.
Panja
Subrata
S
Department of Biochemistry and Biophysics, University of Kalyani, West Bengal, India.
Aich
Pulakesh
P
Jana
Bimal
B
Basu
Tarakdas
T
D002122:Calcium Chloride / Q000737:chemistry*; D003080:Cold Temperature; D004926:Escherichia coli / Q000648:ultrastructure; D006358:Hot Temperature; D008560:Membrane Fluidity; D008563:Membrane Lipids; D018625:Microscopy, Atomic Force; D010957:Plasmids / Q000737:chemistry*; D013499:Surface Properties; D014170:Transformation, Genetic
D008563:Membrane Lipids; D002122:Calcium Chloride