Aeration
Craig
depletion on protein production. It's uploaded under "Aeration
Experiment."
What I did was took a bacterial strain containing a protein I had and
induced protein production at which point I took the bacteria off the
shaker. Uploaded is a picture of a protein gel stained with commassie.
+ refuers to supplying oxygen, which means keeping the flask on the
shaker and - means they are kept still after induction. The optimal
induction time for this protein (from what I have done before) is
about 5 hours, but I also did two more with 15hours of induction just
to see if the protein production takes longer under oxygen-depleted
conditions.
The results show that although protein levels are much lower in the un-
shaked bacteria, there is production. Looking at the relative
intensities of the 5- and 15- induced bands, longer induction times do
not seem to significantly increase induced protein levels but instead
seems only to produce more bacterial protein (the other bands on the
gel). This probably means that most of the induced protein production
is relatively early: either all of the inducer is used up or it gets
degraded or something of the sort. Remember this is induced production
where ample amounts of protein are forced to be produced. The case may
be different if we are using a constitutively expressing plasmid.
Methods:
Used a BL-21 strain containing an insert encoding a protein (app.
33kDa) in pGex vector. pGex creates a GST-fusion protein. Grew colony
for 15hours at 37 degrees on a shaker in 2*YT media (basically rich
media containing an excess of nutrients) +ampicillin.
Diluted the overnight 1/100 into 50mL of 2*YT media and left on a
shaker at 25 degrees until the optical density at 595nm reached about
1 against the media. Added 50microliters IPTG (inducer for pGex) at
which point I took off the - flasks from the shaker. Left for 5 or 15
hours. Centrifuged bacteria at 8000rpm (4 degrees) for 10 minutes,
discarded the supernatent and resuspended the bacteria in 5mL PBS
buffer. Added 5 microliters of PMSF (a serine proteinase inhibitor)
and sonicated for 3*15seconds. Added Triton-X to a final concentration
of 1% and mixed on a wheel for 30 minutes. Centrifuged for 10 minutes
at 12000rpm at 4 degrees. Since I new my protein was insoluble from
previous experiments, I discarded the supernatant (the soluble
fraction) and resuspended in 5mL of TBS buffer. 20 microliters from
each was stained with protein dye (5miroliters) and heated for 10
minutes at app. 100 degrees. These were run on a 8.75% acrylamide SDS
gel and stained with commassie blue.
We'll have to see if we get enough for the bacteria to aggregate, but
I think this does show that anaerobic conditions can be used to
produce protein too.