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fermentation of coli and plasmid stability

Artem Evdokimov aevdokimoz at cinci.rr.com
Tue May 13 22:05:22 EST 2003


My two cents:

Loss of expression of your desired 'stuff' in the absence of selection
pressure will greatly depend on multiple parameters of which at least a few
you cannot control - most notably the toxicity of your gene, metabolic cost
for gene production and plasmid maintenance. You can play around with
changing expression strains, using tight promoters, low-copy number small
plasmids, rec- mutants and related tricks, but if all else fails you will
have to use some sort of selection pressure to maintain the plasmid (by the
way, if the protein is really nasty, the cells will figure out a way to stop
making it eventually, or they will all die and that's not good either).
Since you cannot use antibiotics, indeed other folks have suggested killer
gene controlled expressions - and when you have no choice, you can always
try to license the use of patented technologies that you need to work -
although I disagree with the current trend of patenting everything.
I am not sure if this has been patented or not - you can take a bacterium
and kill an essential gene and at the same time to reconstitute the same
gene using a low-copy number controllable plasmid. Your gene of interest
will also live on that plasmid - so if the plasmid is lost the cells cannot
survive due to the absence of the vitally important gene. This is easier
said than done, because knocking out essential genes in bacteria and having
the resulting bugs survive, even if you are reconstituting the gene with the
plasmid is a big pain in the behind.

By the way, if this method has not been patented yet, it should be - and
some of the royalties can go to me :) Whopee.

A.G.E.

"Ede" <bggfg at gmx.de> wrote in message
news:vjqvbv89ai20ch4h2hv718jts9duopstbl at 4ax.com...
> On Mon, 12 May 2003 10:10:08 +0100, Duncan Clark <junk@[127.0.0.1]>
> wrote:
>
> >Historians believe that in newspost
> ><3EBB385C.6080700 at biotech.NOSPAM.ntnu.no> on Fri, 9 May 2003, Trond Erik
> >Vee Aune <trondaun at biotech.NOSPAM.ntnu.no> penned the following literary
> >masterpiece:
> >>You could try using some kind of partition (par) or killer system (i.e.
> >>hok/sok)
> >
> >I agree fully. Look for a paper in Biotechnology Vol 6 December 1988 p
> >1402-1405 by Kenn Gerdes with hok/sok on a small 580bp cassette.
> >Stabilisation factor of 1000 for pBR322 and 10000 for pACYC, p15 ori,
> >derivatives
> >
> >Duncan
> I`m working in a biotech company and when i use this hok/sok system it
> have to be patent free. somebody have informations
> More about my plasmid: Its a high copy plasmid with an kanamycin
> cassette, but in an industrial process you cannot use antibiotics, and
> this plasmid is to express proteins (whatelse) but the thing is that
> plasmdi free cells overgrow cells with plasmids. So the process is
> shit, but i have to show at the end of the fermentation that most of
> the cells still have the plasmid!!! I hope somebody have an idea
> Ed
>
>





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