Hi everyone, I have a question about bacterial competence. What I understand about bacterial transformation is that they must be competent. So for us to transform our bacteria we must make/buy them chemically or electro competent. But I want to understand how does the uptake really takes place.
I know that for the uptake DNA must be bind to bacterial cell surface, reason why we apply the chemical treatment with CaCl2 (for the bacteria to have a neutral cell surface to which DNA can easily bind and avoid repulsion between membrane and DNA) and then we apply heat shock in order to affect the fluidity of the membrane to permit DNA uptake.
What about electro competent cells? The procedure for making them electro-competent doesn't involve any chemical such as the other procedure, it just involves growing them until a certain phase (I'm omitting the next steps to be more concise) and then applying the electrical shock which creates transient pores on the membranes. But when did I let the DNA binding to the cells? How does this work, it is just the floating nearby DNA entering due the presence of the pores? Does anyone know a good article to read about it or a book.. ?
Thank you very much, sorry for the long post.
The principle of electroporation is by applying an transient electrical field to cells solution to create a transient movement of the charged particles to penetrate thorough the cell membrane. In the instant of electrical field discharge, negative charged DNA molecules will "shot" into the cells on its way to the positive electrode. Since this kind of transformation is to use electrical force to "shot" the DNA into the cells, electoconpetent cells normally are prepared just washing the cells using sterilized water without any salt or charged chemicals. Salt or charged chemicals in transformation mixture can create strong electrical current (electrical sparkling) which will kill the cells.
Thanks for the answer Dr. Liu. In this case basically we are preparing the bacteria for the shock but not for them to bind the DNA ... that's what I understand, am I right? When we prepare the electrocompetent cells we grow them until a certain phase, would this make them more "resilient" to the shock? With the shock we are generating a transient competence for the DNA to enter and transform the cell? When do you say that the bacteria are competent is this process? Thank you! This is a very interesting topic for me.
Yes, that's right, binding of the DNA onto the cell surface is not critical required in electroporation. Although you can prepare the cells at any growth phases, bacteria in early log phase are more acceptable to plasmid DNA (replication) resulting a higher transformation efficiency. The DNA is "electro-shot" into the cells and transient competence is not a commonly used term. All bacteria are more acceptable to plasmid DNA at their early log phase for any transformation and the electrocompetent cells are mainly used to distinguish that from the chemical competent cells.
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