|MadSci Network: Molecular Biology|
Hello, Thanks for sending in your question. Due to the wording, I'm assuming that you are referring to the transformation of Escherichia coli bacteria by plasmids and will be answering accordingly. If you wanted more general information, then please get back in touch. You asked if there were any "human-made" variables apart from altering the heat-shock and calcium chloride concentrations that could affect a bacterium's ability to transform. Basically, the three steps we can consider are: the growth of the bacteria, the conditions under which the bacteria are made competent (i.e. become able to take up foreign DNA), and the step where bacteria and plasmids are added together. Growth of the bacteria in a flask with a good surface to volume ratio and shaking is important, as is collecting the cells during the log phase. The presence of Magnesium ions, (10 - 20mM) in the growth medium enhances transformation efficiency. A number of components act to make E. coli cells competent. These include a divalent alkali earth metal, (e.g. manganese or calcium), a monovalent cation, (e.g. rubidium or potassium), dimethyl sulfoxide, dithiothreitol and cobalt. Except for the cobalt, these can all be replaced by factors that act in a similar way. For example, the monovalent cation could be rubidium or potassium, or even sodium, dimethyl sulfoxide can be replaced by dimethyl formamide, and so on. Certain replacements will effect transformation efficiency. For example, I have read that manganese is more effective in increasing transformation efficiency than calcium. The bacteria and plasmids are usually added together under cool conditions, (0 to 4 degrees Celsius), and most people employ a heat-shock technique as you mentioned. Personally I always used a temperature of 42 degrees Celsius. I have heard that the duration of the heat shock can be important to transformation efficiency although I never looked into this. (I always used 90 seconds.) Of course, the length of the heat shock should depend on the thickness of the tubes your bacteria are in. The size of the plasmid itself can affect the transformation efficiency; the larger the plasmid, the lower the efficiency. I hope this has begun to answer your question. There was an extensive study on the transformation of E. coli with plasmids that might be worth looking at if you have access to the journal "Journal of Molecular Biology". The reference is: Hanahan, D. 1983 Studies on Transformation of Escherichia coli with Plasmids. Journal Of Molecular Biology, 166, pp. 557-580.
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