Transducing rec+ to recA1 E.coli with P1

Richard E. Showalter show at agouron.com
Tue Feb 14 19:10:11 EST 1995


In article <653237897wnr at genesys.demon.co.uk>, Duncan at genesys.demon.co.uk wrote:

> Hi Folks,
> 
> We are having serious problems trying to transduce a recA1 E.coli 
> (Invitrogen TOP10) to recA+ using P1 lysates of JC10241 (from 
> B.Bachmann @ CGSC). JC10241 has Tn10 (TetR) in srl-300 right next 
> to recA. The bug is definitely tetR but it just refuses to go across 
> to TOP10. This is the standard strain for transducing recA+. The 
> lysates appear to be OK, I even did a 250ml lysate and concentrated 
> it with PEG/NaCl etc to 1ml. Still no go. We can transduce other 
> markers from different E.coli P1 lysates into TOP10 although they do 
> go much better into say RR1. I particularly want to use TOP10 'cos it 
> delted for lacz and mcr restriction systems. 
> 
> All idea appreciated even more so if you have transduced recA+ and 
> have a lysate!
> 
> Many thanks
> 
> Duncan
> 
> -- 
> -----------------------------------------------------------------------------
> My mind's made up. Don't confuse me with the facts!


RR1 is recA+ which is why it can be transduced with greater efficiency,
since your tranducing DNA is ds linear chromosomal DNA from the host of
the P1 lysis, it cannot persist very well in a recA- host such as TOP10. 
You did not say weather or not you are getting the TetR marker to
transduce into any of your strains (RR1) or TOP10.  I would test for this
first.  Remember that TetR is induced in the presence of tetracycline
itself so your tranduction frequency will be reduced compared to other
markers.  You can help this induction by giving your tranductions a slight
pregrowth ~30-60min in the presence of autoclaved chlortetracycline.  This
will induce the TetR marker without killing your cells.  Also, how are you
checking for recA+ phenotype?  UV resistence or resistence to DNA damaging
agents such as MMS(methyl methanesulfonate) or
NQO(4-nitroquinoline-1-oxide).  I prefer MMS because UV is hard to control
during a selection.  100ul of a 2% solution spread onto a petri plate
should select for recA+ E.coli over recA-, you milage may vary.  Are you
using P1clr100CMr to make your lysates and transducing at elevated
temperature to prevent P1 lysogeny?  As an aside, you do not want P1 in a
transformation host strain because P1 has it's own
restriction/modification system and will reduce transformation efficiency
when transformed with unmodified DNA.  With the CM marker you can directly
test for non-lysogens and because this P1 is temperature sensitive, you
can reduce the chance of getting a lysogen in the first place.  I am more
familiar with cloning recA from undomesticated marine bacteria and
mutating it out than I am with it's replacement but I would be happy to
answer any questions that I can.

-- 
Richard E. Showalter             "Hey Hey Hey Hey it was
Senior Associate Scientist        the DNA.  Hey Hey Hey Hey
Agouron Pharmaceuticals, Inc.     that made me this way."
show at agouron.com                  Queen-Shear Heart Attack



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