MadSci Network: Molecular Biology

Re: What variables may affect a bacterium's ability to transform?

Date: Wed Dec 6 04:41:35 2000
Posted By: Bela Tiwari, Staff, Bioinformatics Centre, Oxford University
Area of science: Molecular Biology
ID: 972424182.Mb


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 

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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