Expert: Jeff Buzby - 11/4/2003

Question
Hi,

I did this lab for one of my classes (2nd year university bio lab methods class to be specific)...I had 2 types of E.Coli bacteria - S/6/5 which is a wild strain, and JM101 which is a lab strain.  What I was doing was seeing if one could take up a plasmid better than another (I used a pretty small one...pGEM).  I thought that the lab strain would do a better job since it was modified for lab use, but I was wrong - the wild strain uptook it about 3 times more.  

I was wondering if you could suggest any reasons for this happening.  My guess right now is that since it's unmodified, maybe it's just more susceptible to taking up plasmids since it isn't being toyed with as often?  I actually have no idea whatsoever.  Or maybe since the plasmid was for ampicillin resistance, and I put these bacteria on ampicillin containing plates, maybe the wild strain just adapts better to antibiotics and didn't take up a plasmid at all?

Thanks so much if you can offer any help.

Answer

Dear Sarah,

The results of your experiment are a little surprising, but perhaps not so much if we consider the properties of typical laboratory E.coli strains.  Ca+2-mediated DNA uptake generally occurs @ a fairly high frequency for most E.coli strains.  Consequently, ease of transformation is not always the primary concern when selecting for a general laboratory strain.  Some other genetic properties that are often of greater importance are those that can effect the stability of cloned DNA inserts in plasmid vectors & those involved in screening transformants.  These include genes encoding for nucleases & recombination factors.  Strains with mutations in these genes can improve the recovery of intact, cloned, DNA inserts, but they might not grow or transform as well as their wild-type counterparts, as one might expect.  Transformant screening also often requires the presence of additional genes on fairly large episomal F-factors, which can be somewhat stressful, as well.  Finally, strains that have been subjected to genetic manipulation & propagated for long periods under laboratory conditions can accumulate a variety of additional, uncharacterized mutations, the effects of which would be unknown.  So your 1st hypothesis regarding the "unmodified" nature of the wild-type E.coli was basically on the right track.

You might want to look into the genetic background of JM101, which should be readiliy available in a molecular biology lab manual, or from the supplier's product info. sheets or catalog (e.g. the Appendix of a Stratagene catalog), to see how it might contribute to your experimental results.

Thanks for the interesting question,


Jeff Buzby, Ph.D.
CHOC Research Institute
AllExperts Molecular Biology