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fermentation of coli and plasmid stability

Ede bggfg at gmx.de
Wed May 14 15:24:01 EST 2003


On Wed, 14 May 2003 03:05:22 GMT, "Artem Evdokimov"
<aevdokimoz at cinci.rr.com> wrote:

>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.
 I will try this and if i write a patent i will make some notes in the
acknowledgement ;-)))
Thanks for your ideas
Ed



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