Biotech Lab

Meeting 2
Vanessa Hernandez

What
we've
done

Streak Koci
Plates
As we continue to experiment in
other projects we continue to
streak bacteria with the Koci IL8
protein to continue to have fresh
bacteria.
Having fresh bacteria will better
guarantee that our experiment
quality when we reach the time
to do the Koci protein purification
and assay.

Essentially we...
Protein
Manufacturing
Process
Modeling
using GFP

PGlo
Liquid
Culture

PGlo Liquid Culture

Pick Colony
To make a liquid culture, we first took a isolated
colony from a fresh bacteria pGLO plate and placed
it inside a culture tube containing 2mls of
LB+Antibiotic. We labeled the tube with all partner
initials and the date.

Then we placed the tube in the shaking incubator at

37C until the culture reached an OD (600nm)
between 0.6-1.0. Then we placed the culture tube at
4C overnight

PGlo Liquid
Culture
Expand Culture
Removed the 2ml starter culture from the refrigerator.
Vortexd the culture briefly to ensure all the cells are
suspended. Pipetted the cells out of the culture tube and
into a 1.5ml centrifuge tube.
Centrifuged the cell briefly to microfuge (full speed for 30
seconds) to pellet the cells. Resuspended the cells in 1ml of
fresh LB media.
Added 0.5ml of resuspended cells to each of the 2 125ml
flasks containing 25mls of LB+Antibiotics. Labeled flasks
with partners initials and the date. Place both flasks in the
shaking incubator at 37C until the culture reaches an OD
(600nm) between 0.6-1.0.

PGlo Liquid Culture

Induce Expression
Took out flasks from the incubator. Removed 1ml of
culture and measured the absorbance to ensure
culture density was between 0.4-1.0 (OD, 600nm,
0.6 recommended).
Labeled one flask Control and one Induced. Added
ARA to the induced flasks to a final concentration of
1.0mM (259ul IPTG stock)
Returned both flasks to the incubator for an
additional 4 more hours.

PGlo Liquid Culture

Harvest and Lyse
We we spun down and washed our tubes with TrisHCI.
We tried to lasagne our bacteria using Rizwana's
protocol but we failed. Then we added lysozyme
from bio-rad and froze our samples again.
Then we spun it down again and discarded the
pellet.

HIC columns
Purifying our Protein
HIC columns stand for hydrophobic interactive
chromatography.
These columns help purify the protein because they
have sticky micro-beads that the proteins can stick
to when trying to purify it.

HIC columns
Purifying our Protein
This journal entry visualizes and describes the
process we did with the HIC columns in order to
attempt to purify our protein.
We used 4 different types of buffers:
1.
2.
3.
4.

Equilibration Buffer
Binding Buffer
Wash Buffer
Elution Buffer

Look at the journal entry to find the exact functions

of each buffer.

PAGE RESULTS
Running Gels
In order to find out what exactly we had in our
samples we had to run a gel to scan our proteins
present.
While running the gels we discovered that the
coomassie blue is old, the gels are old, and the
kaleidoscope is old.
This is the reason our gels didn't come out more
visual to see our protein better.

Page results picture

edit
Before

After

PAGE RESULTS Conclusion

Final Conclusion
As you can see on this picture the kaleidoscope is
more visual and we can see that there is GFP
protein present.
However the protein is not pure which leads us to
the conclusion that the HIC columns didn't do a
good job at purifying the protein.

Page results conclusion

Final Conclusion
We had many proteins but we will learn from our
mistakes and not commit the same errors.
Personally I can really say we have grown as a team
working together and learning from our experiences.
Great Job Team!

What's Next???
Next Steps
Next we will prepare to do an assay.
Continue to steak fresh Koci plates.
Prepare for Koci, Rizwana, and Luca's visit.
Continue to work as a team to improve our
performance.
AND DON't FORGET TO WRITE STUFF
DOWN IN YOUR LAB NOTEBOOKS.