fermentation of coli and plasmid stability

Ede bggfg at gmx.de
Tue May 13 16:28:49 EST 2003


we checked the stability by plating out diluted cultures on selective
and non selective media. How you check your stability.
Ede
>Dear Ede,
>
> I may sound stupid but are you really losing plasmids (I mean
>experimentally proven or your are just worried about the possibility.)
>
>With my(though limited) experience with E.coli with a set up similar
>to yours(industry with expression of protein), we actually have
>observed very little or no plasmid loss after fermentation in absence
>of selection pressure. Though I know it differs from one expression
>system to other. I am not sure it is a very common phenomenon with
>high copy no. plasmids.  EK or DUNCAN  to comment please.
>
>Some people feel that high growth rate causes loss of plasmids as they
>are not able to partition properly in very fast growing cells. Though
>I am not sure about it's implication in high copy no. plasmids. 
>Reducing the growth rate in such a case should obviously be tried.
>
>Anyway discussion continues
>
>
>S.Ballal
>Indus Biotherapeutics Ltd.
>Ahmedabad, India
>http://www.indusbio.co.in
>
>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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