P1 transduction problems
chrisb at hgu.mrc.ac.uk
Thu Feb 18 07:12:41 EST 1999
, David Alexander (dalex at NEXUS.MICROIMM.MCGILL.CA) wrote:
: Hello Netters,
: I am attempting to transduce a non-essential gene containing
: a TetR marker from E. coli MG1655 to MC4100.
: The P1 vir lysate made on the MG1655 strain with my gene/TetR
: is of very high titer (believe it or not, around 10 ^ 16)
: However, I have yet to see a TetR transductant.
Near the bottom of http://www-micro.msb.le.ac.uk/224/Phages.html you
can read about M13 and similar phages:
> These are temperate (i.e. non-lytic) phages - establish permanent
> infections without lysogeny. ~300 particles/cell/generation are
> produced; titres in infected cultures up to 5x10^12/ml (~150mg/ml) -
> good recombinant vectors.
For M13 phages, a titre of 10^16/ml implies a density of 2 x 10^3 x
150mg/ml = 300 g/ml. Bearing in mind that P1 is well over 10 times
bigger than M13, you can see your claimed titre would give a
"suspension" with a density of > 3000. For comparison, the density of
plutonium is 19.35.
It's therefore CERTAIN you have MUCH less P1 there than you think. P1
plaques are tiny and easily swamped by contamination with virtually any
other phage. If I were you I'd try to get a better lysate, or assume
you have a low titre lysate and repeat the transduction with undiluted
Also, if the Tc marker in the incoming DNA is on Tn10, it doesn't hurt
to prevent the tetR product from repressing the resistance gene --
allow cells to express after transduction for at least an hour in the
presence of sub-inhibitory amounts of tetracycline (say 1/100 normal
concentration) before plating out.
Chris Boyd | from (but not \ MRC Human Genetics Unit
Christopher.Boyd at hgu.mrc.ac.uk | on behalf of) / Crewe Rd, Edinburgh
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd EH4 2XU, SCOTLAND
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