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Bloopers

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Now what did we learn??? hmmm...

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1. Prepare 2.0 litre of Luria-Bertani Medium (LB),
which is used as both seed-culture and fermentation media.

Recipe of LB (Luria- Bertani Medium)
1. Bacto-trytone 10g
2. Yeast extract 5g
3. NaCl 10g
4. dH2O 1000ml
5. pH 7.5

In order to get ready the medium for usage, at least 2 liters have to be prepared.
100ml of the medium is transferred to a 500ml shaker flask and
the rest was dumped into the bioreactor.
Both the flask and the fermenter are autoclaved for 20 minutes.
Once they have cooled to 50°C, ampicillin is added to the final medium and kept at 4°C.
(The reason why the ampicillin [it is an antibiotic] doesn’t kill the bacteria that
you’re trying to grow is that it has a the ability to resist the ampicillin.
Thus, the ampicillin would only kill any OTHER bacteria that you’re growing)

Indeed, these cells that we grow are very much like
the little toddlers that bring us so much joy and frustration.
The same way we have to throw their milk bottles into
steaming vats of boiling water, the bioreactors have to be autoclaved
(this means they’re put into a really, really hot place. I mean REALLY hawt.)

4. Add ampicillin to a final concentration of 100mg/ml
(both seed and fermentation media)
after the broth has cooled to below 50°C.

2. Bioreactor Preparation Steps

1. Calibrate the pH electrode using electrode
using standard buffer solution

Calibrate it by aerating with nitrogen.

7. Connect the addition lines to peristaltic pumps.
Switch to “Auto” or “Manual” control appropriately.

We start off by making sure the pH electrode is
reading the right reading by using a solution of
known pH and getting it to read the pH value
of the solution. So we actually know the value
of the solution and seeing if the pH electrode
will give the same value when it reads.
This is kinda like how the police like to pretend
they don’t know anything and ask the criminal questions,
waiting for him to make a slip so
they can corner him. Oh, wait…that’s out of topic….

Anyway…

The next step is to install the different probes,
which are like sensors. The probes stick down from
the ceiling of the fermenter like stalactites, so
we have to make sure the probes are low enough to
touch the liquid, or else they would just be
measuring thin air.

After that, the pump lines for the acid, base and antifoam
are plugged in and the levels of the storage bottles which
they keep these acids, bases and antifoam are checked
to make sure they are sufficient.
(You wouldn’t want the machine to be pumping in air
when its supposed to be pumping in antifoam, would you?)
And once the other accessories like the exhaust condensers,
air inlet and exhaust filters are installed,
we are ready to autoclave the fermenter…..well, almost.

All of the cables are disconnected from the fermenter
except the temperature probe, which is autoclavable.
The silicone tubings are all clamped down except
for the exhaust filter. The filters and sockets were then covered
with aluminium foil to protect them from condensing moisture.
Finally the whole thing is autoclaved with steam
at one hundred and twenty one degrees
(No, I wasn’t kidding about the heat ;p ) for 20 minutes.

Once this is done you can fix everything back,
calibrate your pO2 probe, and hook up the addition lines
to the peristaltic pumps, you’re ready to put stuff inside!

Note: Peristaltic pumps are pumps that work just
the way your esophagus/GIT works when it
pushes your food all the way down. i.e. with
that little wavy motion of muscular contraction.

Another note: Please for goodness sake,
don’t use hydrochloric acid
for your pH correction agent because it has chlorine ions.
And chlorine is BAD because it’s what
they put into swimming pools where kids pee a lot to KILL bacteria.
So unless you’d like your fermentor to be a graveyard,
don’t use HCl !!!!!!

and arabinose 0.2% and incubate it for 24hrs.

3. Streak colonies of pGLO transformed E.Coli from LB/Amp/Ara plate,

4. Place flask in shaking incubator and incubate at 32°C
for 24 hours or at room temperature for 48 hours.
This will be used to inoculate the fermentor for scale-up fermentation.

of 100ug/ml and of 0.2% respectively.

2. And the control parameters was set up as follows:
Temperature - 32˚C
pH - 7.5
Stirring speed - Minimum 10%, maximum 90%
Control to AUTO
pO2 Set Point - Set Point 20%, Control to AUTO
Stir to CASC & AIRFLOW to CASC
Airflow - Minimum 25%, Maximum 100%
*Optimal GFP folding and fluorescence occur at 32˚C and below
Luckily for us, the monitoring of the fermenter is done for us
by machines. You simply have to set the ranges that would
allow the bacteria to grow and the machine would keep
all the Oxygen concentrations, pH levels,
stirring speed, airflow and basically everything for you.

Inoculated the fermenter with 100ml of seed culture [5% of fermentation medium volume]. Fermentation was left to be carried out for 24 hours at the conditions mentioned. Before inoculation was carried out, a 10ml blank sample was taken, Then took a sample of 10ml after every hour after. The fermentation broth harvested after 24 hours fermentation. Placed 10ml of culture into a sterile and disposable test tube.

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So now, let us have Professer Nest to give us a explanation.

Bear with him, he's kinda dragy.

10mL of the culture broth was collected into a tube
for the whole experiment. Centrifuged the cells
at 10,000 rpm for 5 minutes which separates the cells
from the liquid broth and forms a pellet at the bottom
of the tube due to it being denser.
Since the liquid broth is less dense,
it constitutes the supernatant.
The supernatant is then transfered into a fresh new tube.
Both tubes were observed under the ultraviolet (UV) light
for product to confirm the results obtained.

Method 1: Use of Enzymes
Resuspend the pellet in 500µl of TE buffer of pH 7.5
with the use of a micropipette.
Ensure there are no visible clumps.
Add to drops of lysozyme into the resuspended cell pellet.
The lysozyme breaks down the cell wall, and hence,
releases the proteins within.
Allow proteins to act for 15 minutes.

Method 2: Freezing and Thawing
Place the tube in liquid nitrogen till
the contents are frozen. Thaw the tube in warm water.
Repeat the cycle of freezing and thawing twice more
to ensure complete disruption of the bacteria cell wall.

The cycle of freezing and thawing adds mechanical stress
to the cell wall, as the cell water content expands when frozen
and contracts when thawed.

Method 3: Sonication
This process is whereby ultrasonic waves are utilized
to cause cell disruption under the vibration pressure.
(Protective ear muffs must be worn when performing experiment)

Carry out sonication by puting it on ice
for 4 cycles of 25 seconds
with 10 seconds rest in between sonication cycles.
Centrifuge the tube for 20 minutes at 10 000 rpm.
Separate the supernatant and pellet.
Resuspend the pellet using 400µL of TE buffer.
Observe the tube under UV light to confirm the product.

Stage 2: Purification
Gel Filtration Permeation also known as
Size Exclusion Chromatography
will be performed to purify the extraction.
This method uses a column of polymer gel resins (Sephadex G75).
Due to the resins containing small pores,
small molecules will be able to diffuse through.

As a result, as the extract is poured into the column,
the bigger molecules will flow through faster,
as the smaller molecules spends more time diffusing
into the pores of the gel resins.
This allows the different molecules to be separated by size.

9 tubes are prepared and labeled.
2ml of ammonium bicarbonate is added into
the tube labeled “blank”. The buffer in the tube
is allowed to drain until it reaches just above
the gel bed, where the supernatant from the
isolation part is added in. Fractions of 2ml
of the drain are collected in the 8 tubes.
Ammonium bicarbonate is added
constantly to prevent the gel from drying.
The fractions collected in the 8 tubes are
subjected to Spectrophotometry to get the
absorbance readings at 476nm
(wavelength where GFP absorbs well).
We use ammonium bicarbonate as the blank
to standardize and compare the absorbance values
with other fractions. We use gel filtration chromatography
to fractionate based on size the proteins in
a cellular extract. Its principle is that the
bigger molecules will flow through the column
faster without diffusing into the pores while
the smaller molecules get diffuse and interact
with the pores of the gel resins. Note that the column
should not allow being run dry; little cracks and
channels are formed when the column runs dry,
and separation of proteins is greatly compromised
as a result. From the graph above, it clearly shows
that fraction number 2 and 3 have the most abs values.
This is because the big florescent molecules elude
quickly into both fractions and because
the size of the molecules is big, hence they
emit more florescent light. As for fraction number 1,
we believe that it did not show one of the most abs
values as the GFP has just started flowing down
and some of the molecules require some time to
flow the column as there are beads within
that are obstructing their flow. Another reason could be
other protein molecules with a size bigger than
the pore size of the beads are competing to flow
through the column with the GFP molecules.
From fraction 4 onwards, the abs values decrease
as the smaller molecules, like other proteins,
diffuse out from the beads and eluded. Gradually,
the number of fluorescent molecules decreases
with each fraction collected as most of them are
already collected at fraction 2 and 3. Therefore
only few of those are left to emit light. At fraction 8,
the abs value is almost zero, and we therefore
conclude that all the GFP had been eluted and
collected in the 8 tubes. From the results, we can
say that we have quite a pure preparation of GFP
as the protein of interest is well separated with
rest of the unwanted proteins.