Q: ColE1 and p15A incompatibility

Wolfgang Schechinger marsupilamiwolfgang at web.de
Wed Jun 19 09:33:00 EST 2002


Dear Lee, 


the actual plasmid copy number might reflect the antibiotic concentration
since those bacteria might have optimal growth that have the optimal gene
concentration for each antibiotic. 


Assuming the number of protein molecules is a function of gene number
(more 
accurate: gene activity, since promoters and transcription factors also
play a 
role).<FontFamily><param>Symbol</param>

<FontFamily><param>Courier New</param>If a resistance gene yields a low
enzymatic activity, there must be a higher 
number of enzyme molecules to make the cell survive a certain antibiotic
concentration than if the enzymatic activity was high.


As long as you are within the linear dose/response curve of an antibiotic,
the 
antibiotic concentration should exert a selection pressure and thus favor
those cells who have an optimal concentration of resistance gene i.e.
plasmid.


When you use two different antibiotics, your system will optimize the 
concentration of each plasmid. Thus it may happen, that you have a lot of
one 
resistance gene product and a little of the other and probably also
different 
plasmid concentrations. <FontFamily><param>Symbol</param>


<FontFamily><param>Courier New</param>To find out if all this is
practically relevant, my hypothesis should be easy 
to prove in a simple experiment:<FontFamily><param>Symbol</param>


<FontFamily><param>Courier New</param>Grow your bacteria starting from one
starting culture with different 
antibiotic conditions, e.g. 10:1, 1:1 and 1:10 relative to the initial 
concentration. Then monitor the concentration of the individual plasmids
at 
different times and also record the OD for obtaining a growth curve and
check 
if you can see a difference. 


Probably you may assay the concentration of each plasmid by simply running
0.1 
to 0.5 ug of a miniprep on an agarose gel (ore use QPCR to obtain more
exact 
data).


If necessary, you then also may monitor the concentrations of your
proteins of 
interest for further optmization since there are other factors present
(like 
protein half life) that will influence your system.


Best regards and good luck,


Wolfgang<FontFamily><param>Symbol</param>


<color><param>7F00,0000,0000</param><FontFamily><param>Courier
New</param>> Hi,

> 

> Actually I'm trying BL21(DE3) Cp strain (Stratagene, contains

> rare codon tRNA vector which is chl resistant) and pKY206

> (tet marker) at the same time. They are derived from pACYC184

> which has both tet and chl markers. As I don't have the map

> of these plasmids, I'm not sure whether the other marker

> is crushed or not. Anyway, I have a transformant and am going

> to miniprep it to check if the transformation was successful.

> 

> I just wanted to know the mechanism of incompatibility.

> Do you have any idea? Could you tell me more about the titration of

> antibiotic concentrations?

> 

> Regards,

> 

> Lee

> 

> > Dear Lee,

> >

> > You could get problems due to different replication speed/rates of the 

> > two plasmids, possibly requiring careful titration of antibiotic 

> > concentrations.

> >

> > Did you already try it? What results did you get?

> >

> > Regards,

> >

> > Wo

> >

> > > I have two plasmids that contain p15A replication origin

> > > but have different antibiotic markers (chl and tet).

> > > Are they still incompatible? If so, why?

> > >

> > > Thanks,

> > >

> > > Lee

> > >

> >

> >

> > ----- Dr. Wolfgang Schechinger E-mail hubahopp at gmx dot de 

> 

> 

> 


-----
Dr. Wolfgang Schechinger
E-mail hubahopp at gmx dot de

---




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