Day 5 - Monday - 5/11/2007

LAST DAY OF PRACTICAL! No more BPT practical yeeehaw! We’re not saying that we didn’t enjoy the practical. You bet we did! It was super fun especially those waiting times. But hey, no one’s complaining. One less practical per week also equals to one less lab report to worry about. 8)

Okay so this was how the practical went that day:
It was a loooong practical, more than 3 hours. Today’s practical we had to complete 3 stages in order to harvest the final product: Isolation, Purification and Analysis,

Firstly, we measured 10 ml of the culture broth in a tube..

Then we obtain the cells by centrifugation at 10 000 rpm for 5 minutes.. (please pretend you see 5 mins instead of 4 mins because we were 1 min late to take the photo haha)

So, we pipette the supernatant out..

..and observe the two tubes under UV light. Oooohhh aaaaahh.

Stage 1: Isolation

This Green Fluorescent Protein (GFP) that we are working with is an intra-cellular product; hence the bacteria cells need to be lysed first to release the protein. In order for that to happen 3 methods was done.

Method 1: Using Enzymes
The pellet was resuspended in 500µl of the TE buffer of pH 7.5 using a micropipetter until there are no visible clumps. Then, two drops of lyzozyme was added to resuspend cell pellet using a disposable 1 mL plastic pipette. Later, the enzyme was allowed to act for 15 minutes.

Did we just hear “wait for 15 minutes”? Yes we did! So this is what we do to spend the 15 minutes:
Thing-to-do-Number-1: Bite Nic’s head!

Thing-to-do-Number-2: Blow your gloves!

Okay back to the practical…

Method 2: Freezing and Thawing
After that, the tube was placed in liquid nitrogen and was thawed in warm water repeatedly. The cycle was repeated for 2 minutes to complete the rupturing of the bacteria cell wall.

Method 3: Sonication
This is the last stage of the isolation process of our product. (THANK GOODNESS!)
Here, the poor cells are exposed to ultrasonic waves which will cause the cell membranes to be disrupted under vibration pressure and release its cellular contents (GFP, which is what we want.) The cycle is done in ice for 4 times, each lasting 25 seconds with 10 seconds rest in between the sonication cycles.

After cell disruption, the contents of the tube were spun in a centrifuge for 20 minutes at 10,000 rpm.

Yes you heard it right again. 20 minutes of waiting! So what shall we do now?? Two choices: You can either act cute like Yamon or act ~~retarded~~cold like Filza.

After 20 minutes...

The pellet and supernatant were separated and resuspended using 400 µL of TE buffer. They were then observed under UV light.

Look at that difference! The supernatant is now the fluorescing under UV light. WHY?!....That’s because the cell walls are now ruptured and its cell contents, the green fluorescence proteins have escaped into the surrounding medium.
This marks the end of GFP isolation process. I wonder what will happen to the cells if we sonicate them too long. Any guesses? Heh. So much for us being villains and lysing the cells! Sorry cells...

STAGE 2: PURIFICATION

Gel Permeation Chromatography or Size Exclusion Chromatography is the method of purification using a column of a polymer gel resin. The resins contain very small pores in which the molecules that are small enough can diffuse within. Hence, when the extract is poured into the column, the large molecules will flow through the column faster whereas the small molecules will spend more time interacting and diffusing into the pores of the gel resins. This achieves separation of the different molecules by size.

The column was drained carefully into a waste beaker until the buffer is just even with the top of the gel bed. A pipette was used to transfer cell-free extract to the top of the gel bed by gently swirling the pipette around the inside edge of the column.

Eight test tubes were labeled and placed in a rack. The Blank wad filled with 2.0 ml of ammonium bicarbonate. Fractions are removed by placing a test tube under the stopcock where the buffer is collected in the test tubes. Each test tube was filled to the 2.0 ml mark before moving on to the next test tube..

50mM ammonium bicarbonate buffer is added to the top of the column while the fractions are taken.

STAGE 3: ANALYSIS

Before we can measure the absorbance value, we have to transfer the solution into the cuvettes. ~DUH

Use the pipettes…

Aiyah so slow! Lets just pour it in..

The absorbance readings were then taken using the spectrophotometer set at 476nm. Where at this wavelength, the GFP is strongly absorbed and gives out its usual fluorescence.

Our Absorbance Readings:

Fraction	OD₄₇₆
Blank	0.0
1	0.014
2	0.080
3	0.032
4	0.027
5	0.015
6	0.010
7	0.001
8	0.012

According to the lab manual, we’re supposed to comment on this graph. So here goes nothing:

From this graph, we can see that Blank, which is ammonium bicarbonate, was used to zero the spectrophotometer.
Fraction 1 has a relatively low OD reading as the sample was just added. Therefore, only certain amount of the sample was able to reach the end of the column.
Fraction 2 has the highest OD reading and is also the peak of the graph. This tells us that a high amount of light was absorbed from this fraction which resulted to the high OD reading. This is because of the high concentration of GFP present in that test tube.
As the fraction goes on from Fraction 2 to Fraction 8, it can be observed that OD reading continues to drop steadily. This shows that the amount of GFP decrease with each test tube.
Also according to the lab manual, we’re supposed to answer a question 2. So here goes again (for mark’s sake):

Answer to qn 2:
The protein with a M_r of 50,000kD will be eluded in a fraction after the GFP because it is heavier than the GFP therefore it will tend to move slower and thus require more time for it to be eluded in the cell free extract.

Finally from this practical, we’ve learnt certain points about fermentation:

Learning points about fermentation in general.

The simplest definition for fermentation is the conversion of carbohydrate into an acid or alcohol. This can also be done by providing nutrients to microorganisms and obtaining their useful by-products. We all know that the fermentation technology is one that has been carried out long before our time (e.g. making of bread or wine) which all started out with very simple instrumentation. Since then, the process of fermentation has improved greatly with the development of computerized equipment. In industrial fermentation, different systems of fermentation may be used such as batch, continuous and fed-batch fermentation. The type of system used is dependent on the desired product. This experiment has taught us about the different conditions (such as temperature, pressure, pH, oxygen content and etc.) required to maintain production efficiency and techniques to recover the desired products in pure form through isolation and purification.

And so that marks the end of Bioprocess Technology practical! We’re so going to miss the fermenting smell of the lab… NOT! Zank you for reading our blog. We hope you’ve enjoyed it. To end this off, a group photo. Awwww choo cweet...

~Amandy & Wawa & Yamone out!