Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Thu Jul 27 10:13:51 EST 2000
In article <8lph8v$5nq$1 at lux1.biobase.dk>, Jesper S. Pedersen
<jsp at NOSPAMimage.dk> writes
>Dr. Duncan Clark <drc at nospam.demon.co.uk> wrote in message
>news:vdL1ySAz9Cg5EAoC at lineone.net...
>> Titre everything onto a F' minus E.coli and then you should only pick up
>> the Ampicillin phagemid. The helper phage will not grow.
>> I think you will need to read a little more about the system you are
>> working with and strain genotypes.
>F' is necesary for phage infection, right? So how can you titre the phage on
>F' minus E. coli? I think you misunderstood med : Under phage production it
>seems that 50% of produced contain the wanted phagemid and 50% contain the
>helper-phage genome. This seems to be a fact... The question is : How can
>you efficiently 'kill' the bacteria that are infected with a phage
>containing the helper-phage genome - to prevent them from producing new
I need my holiday starting Saturday for two weeks.
My gross mistake. I was thinking things like electroporating mixes of
phagemid and helper phage into F' minus E.coli (which what we were doing
not too long ago for specifically losing the helper). Apologies for the
error and my last comment. A single phage particle is produced which I
think gets kicked out.
We used to do this fifteen or more years ago by transforming M13
mutagenesis reactions into a mutS F' minus E.coli then plating the lot
with TG2. The phage particle goes out of the mutS and infects the TG2 so
you got a plaque that way etc.
Are any of the helpers around supE or supF dependant so you can pick an
appropriately F' genotyped E.coli to passage through etc. Again I think
we used to do something like this with early M13's (from the original
Messing paper in Gene) and making uracil phage by having to actually use
a supF UNG minus E.coli, RZ1032 springs to mind versus CJ236.
Have you tried alternative helpers that preferentially package the
phagemid over themselves. It may reduce the phage titre another 10E2 etc
which may be all you need.
> Another problem is that if both the helper-phagevector and
>the phagemid in some rare cases are packaged into one phage particle the
>bacteria infected with this phage would be able to still produce phage.
With titres being so high would that scenario be rare? Cells plated
within a mm or so of one another may (anyone know?) pick up helper phage
from the next door colony that has this 'rare' event.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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