MadSci Network: Molecular Biology |
One can construct a saturation curve for the transformation of
E.coli, and
it is often a prudent measure for large-scale transformations. It is
valuable to titre the experimental DNA to maximise the colony-forming
potential for each discrete transformation.
What one usually sees is a progressive (linear) increase in transformation efficiency with an incremental increase in the amount of plasmid DNA. This begins to tail off (become non-linear) as the ratio of plasmid DNA to number of cells reaches saturation. The point is to get maximum transformation with minimum DNA - afterall, anyone can take a commercial plasmid stock and transform it into E.coli; I frequently do and think not a jot about optimising the process. However, when you have experimental DNA, you want to use as little as possible, whilst optimising the transformant recovery as much as possible. The aim is to know exactly how many cells you have and how many DNA molecules you need. The level of transformation efficiency will obviously tail off regardless of how much extra DNA to plough into the mix as there is a theoretical maximum number of transformed cells you can have, largely governed by a) the number of cells you have; and b) the method by which they are made competent. This is where determining transformation efficiency and expected XFE values comes in; together with determining the Fc (fraction of viable cells) [See references]. Once saturation has been attained, you simply get double transformants and waste previous DNA. You may also have the issue of competition with foreign or other non-specific non-transformable DNA that occupies sites on the cell surfaces. It has been documented that the quantity of experimental DNA used, the less effect this has - but is not something I have encountered / needed to think about in my work. As far as work with pAMP and pCAN is concerned, I have never worked with, or seen the need for a double transformation with these plasmids. Now in double transformations I have done in the past (assuming that they are compatable plasmids), I have actually noted increased efficiency using them together than each individually - but I could not say whether this is specific to certain plasmids and bacterial genera - or a more general phenominon....It is not generally unheard of, and I'm sure there is a lengthy reason behind it. Rather than write a review on the various theories, I shall point you in the direction of a couple of good reviews on general transformation. Hope you find something useful in this response :0)
Jim Caryl References (in order of usefulness): Hanahan, D. (1983). Journal of Molecular Biology 166: 557- This is a good one to get...He takes you through (step-by-step) the process of designing and implementing a transformation experiment. General reviews on transformation: Hanahan, D., Jessee, J. & Bloom, F. (1991). Plasmid transformation of Escherichia coli and other bacteria. Methods in Enzymology 204: 63- 113. Smith, H.O. & Donner, D.B. (1981). Genetic transformation. Annual Review of Biochemistry 50: 41-68.
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