Re: [DIYbio] Re: GFP/Plasmid Project

On Wed, Mar 14, 2012 at 2:25 PM, Erik Garrison <erik.garrison@gmail.com> wrote:

>
> An impure isolate, with a small number of non-green cells relative to green
> ones, produced no GFP production.  Although this could have been due to
> other issues, such as poor incubation conditions, it seemed plausible that
> non-GFP producing cells out-competed those which did, leading to a result
> which had no observable GFP production.  (I did this.  Beginner's mistake, I
> hope.)
>
> It got me thinking: what exactly is happening to the plasmid sequence in
> these cultures?  The bla gene must not be degraded, or the cells couldn't
> grow on ampicillin media.  However, the GFP gene is not under any kind of
> regulation, and is in effect turned on all the time, so any cell which has
> it should produce some GFP, albeit inefficiently if under poor conditions.
>  It seems that this is a case of selective degradation, as without pressure
> on the production of the GFP, it will be relatively selected against.

The /Bla/ gene might be gone, but the BLA protein may still be around
post-cell death... (of the out-competed transformants)

Seems like your ampicillin concentration was probably just too low,
the initial cells in the area you subcultured were just slow to
grow/replicate, but then the green cells decreased the concentration
of amp even further with BLA, which lead to faster growth for the
wild-type cells and eventual takeover by them.