Shang-Feng
Immanuel
Meena
Filza
Shu Fung
Nicholas
Wahyudin
Wahyuna
Ya Mon
Amanda
Anwar

Yo yo~
Wyd here to keep you up to speed on what's happening right here in our wacky-looking blog!
Woohoo~! =D
First and foremost, I would like to thank everyone that has made this possible
Namely, Mr Ong Chee Meng for deligating us the task of doing this really innovative project
Ms Ang Sheu Ngo who taught us the ropes throughout the practical sessions
The ever-so-helpful lab techs who were always there to aid us whenever we need them
And everyone else in the group~! (Not to worry, I'll introduce everyone a little later)
Woops, I think I forgot something else... Thank you NYP for providing us with all the equipment and facilities we need during the course of the experiments!


Now without further adieu, let me introduce our group... the MB0602 group B!
*drum-roll*...
For your convenience, please refer to the group picture at the top. I'll be introducing everyone clockwise, starting from the bottom right.

Ishwarya Meena aka Meena~chan!
Zin Ya mon aka Yamone (or pikachu =X)
Wahyuna Sulaiman aka Wawa
Amanda Yap aka Amandy
Nur Filza Baharam aka Fil
Tan Shu Fung (aka the Tai tai~)
Immanuel Kwok (I'm-manual aka I'm-Not-Auto XD)
Wahyudin Hassan aka Wyd (eh wait, datz me! ^^)
Chia Shang-Feng aka Ice Man
Anwar Muhd Saleh (eh.. didn't relli think of a nickname for him yet)
Nicholas Tan aka Nicky~ (or the rich !@#$%)


As you can see, each day, different people are allocated the task of blogging their own entry. This is what makes it interesting!
You will be able to see the different touches in blogging, ranging from the super formal post in day 1 (mostly thanks to Nicky.. he's quite boring. =X) to the post filled with lame jokes in day 3 (now you know why they call him the Ice Man...) and the very comical post in day 5!(hurray for Wawa, Yamone and Amandy!)


Anyway, don't forget to tag!
Enjoy ppl~ =)

-Wyd-

Bioprocess Technology Lab

Contents
Overall objective:
To grow and extract green florescent protein

Individual objectives:

Experiment 1

To be familiarized with the parts and components of microbial and mammalian bioreactors

To introduce the basic operation procedure of a bioreactor

Experiment 2

To describe the steps to prepare a bioreactor

To prepare the media for seed culture and scale up fermentation

To prepare seed culture for scale-up fermentation

Experiment 3

To carry out scale up fermentation process to increase the yield of desired protein product

To monitor cell growth and product formation through manual sampling and computer data logging

Part of Bioreactor	Purpose
Motor	To power impeller
Impeller	To spin culture and to mix media. This ensures that there is no settling of cells
Sparger	Introduce air into fermenter so that cells can take up dissolved oxygen
Baffles	Introduce turbulence and for uniform mixing
Inlet Air Filter	To remove all contaminants found in the air
Exhaust Air Filter	To prevent contaminants from being released into the surrounding
Rotameter	To measure air flow
Pressure Gauge	To measure air pressure of fermenter
Temperature Probe	To monitor temperature changes and to maintain optimal temperature for cell growth
Cooling Jacket	To cool down reactor and control the temperature of reactor
pH Probe	To monitor pH level changes and to maintain optimal pH for cell growth
Dissolved Oxygen Probe	To measure the amount of dissolved oxygen so that cells get adequate oxygen for growth
Level Probe	To maintain constant amount of media
Foam Probe	To measure the level of foam produced so that it does not choke up the fermenter system
Acid	Added when pH level is too high to provide optimal pH for cell growth
Base	Added when pH level is too low to provide optimal pH for cell growth
Antifoam	To reduce the foams produced
Sampling tube	To monitor growth of cells
Control Panel	To specify all parameters

Procedure for preparation of equipment, media and culture:

Media for Seed-Culture and Scale-up Fermentation

Today was the first day of our bioprocess technology (BPT) lab. It was quite fun coz we managed to do quite a number of hands on stuff. The first thing that we had to make after the briefing was preparing the media. We were asked to make about 2.0 litres of Luria-Bertani (LB) media. We had to make that first coz the LB media was needed for both the seed-culture and for the fermentation media.




The commercially made LB broth powder, which made our life easier.


Making the media was quite easy as we only had to measure and add the ingredients together. The only challenge was how accurate our measurements were. The recipe was given to us, which was 10g of Bacto-tryptone, 5g of yeast extract, 10g of NaCl, 1000ml of distilled water and maintaining the pH at 7.5. Since the lecturers were nice enough to make our life easier, we only needed to add the pre-mixed water with 1000ml of distilled water. The other ingredients were already mixed for us. Even the overall pH! How lucky was that.




Immanuel and Filza measuring the LB broth powder.




Look at how precise it was. We were suppose to measure 50g of the LB powder.




Anwar and Filza adding the smelly powder into the distilled water.




Anwar making sure that the LB broth was exactly 2 litres.


Anyway, we transferred about 100ml of the media into a 500ml shaker flask and the remaining 1.900ml of the media into the 2-litre bioreactor. Both the shaker flask and the bioreactor was autoclave at 121^oC for 20 minutes. We were lucky (yet again) that the lab technician helped us to do this part of the procedure and even assists us in adding the ampicillin to a final concentration of 100ug/ml (for both flask and fermentation media), after the broth was cooled to about 50^oC.
The media preparation is complete so the media was kept at 4^oC till inoculation.




Wahyuna excitedly poured the LB broth into the 2 litre jar.




Our LB media all done! (looks like urine though~)


Next we had to prepare the bioreactor. This was a little more complicated. However, we had the assistance of the lab technician to help us in majority of the preparation work. He explained to us what had to be done. So, first thing first, we had to calibrate the pH electrode using the standard buffer solution. After calibrating it, all the probes were installed like the probes for pH, pO₂, foam and level. The foam and level probe was also adjusted. When the probes were placed correctly, the addition agent lines for acid, base and antifoam were connected. The levels in the storage bottles were also checked and it was brought to our attention that hydrochloric acid is so not suitable as a corrective agent for pH.

The next step was installing all the other necessary accessories such as the exhaust condensers, air inlet and exhaust filters and manual sampler unit were.
After installing, we checked the water jacket to see if it is filled with water.

When all the installing was completed, we prepared the bioreactor for sterilization. To do this, we had to disconnect all cables except the temperature probe, which is also autoclavable. We also had to clamp all the silicone tubings’ except for the exhaust filter and female STT coupling of sampling unit, cover all the filters and sockets with aluminum foil to protect from condensing moisture. Finally when we finished all the preparation, the bioreactor was autoclaved with steam at 121^oC for 20 minutes. The pO₂ probe was polarized for 6 hours and calibrated by aerating it with nitrogen.

Finally, the final two steps in preparing the bioreactor. First, was having to connect the addition lines to the peristaltic pumps. Of course we made sure that the pumping direction was correct. Last but not the least, the switch was changed to "Auto" or "manual" where needed.

NOW’s THE GROWING OF OUR BABIES part!
Transformed E.coli with the GFP protein had to be grown up in a media plate with Amp and Ara. WHY?! The reason is partly to remove other types of bacterial strains growing in the media before streaking, or removing those asses that were transformed with GFP. That will be Ampicillin, Amp’s job! After streaking, it’s time to let them grow and they can be transferred to a larger flask the next day.


Just FYI:

1. On media preparation:
a. Bacto tryptone is a protein source for the growing bacteria like E. coli.

b. Yeast extract is used to provide the nutrients for the bacteria growing.

c. DH₂O is used to dilute the solution and to dissolve all the components.

d. Ampicillin is an antibiotic used to prevent other bacteria besides the targeted bacteria from forming.

e. Ampicillin is only added after autoclaving as the heat from the autoclaving can deactivate the antibiotic.


2. On equipment preparation:
a. Calibration of the pH probe meant that the probe reading had to be adjusted for optimal and accurate reading of pH during the run.

b. Hydrochloric acid is not suitable as a correction agent for pH because it is a strong acid and by adding a small amount of acid, it can create a big change in the solution.

c. A voltage is applied across the electrodes of the Dissolved Oxygen probe. Silver ions are oxidized at the anode producing silver chloride and electrons. Oxygen is reduced at the cathode accepting electrons and producing hydroxide ions. The current generated by the chemical reactions is conducted through the filling solution. This current is proportional to the oxygen concentration and is converted by the meter to a dissolved oxygen reading. Polarization is the term used to describe when the probe has reached equilibrium, and is able to produce stable, reliable measurements. For the probe to be polarized, voltage must continually be applied to the probe, allowing the oxygen reaction to proceed. When voltage stops, the reaction no longer continues and the probe is no longer considered polarized.

d. A peristaltic pump is a type of positive displacement pump used for pumping a variety of fluids. The fluid is contained within a flexible tube fitted inside a circular pump casing (though linear peristaltic pumps have been made). A rotor with a number of 'rollers', 'shoes' or 'wipers' attached to the external circumference compresses the flexible tube. As the rotor turns, the part of tube under compression closes (or 'occludes') thus forcing the fluid to be pumped to move through the tube. Additionally, as the tube opens to its natural state after the passing of the cam ('restitution') fluid flow is induced to the pump.


3a. The arabinose serves a GFP inducer. It is an inducer that will induce the cell to produce more GFP when the cell comes into contact with the arabinose in the agar plate. The cells in this study uses arabinose as a carbon source, and if the amount of arabinose available to act as an inducer was low, the production of GFP would be lowered as well.

3b. The environment and materials in the flow hood had to be swabbed with ethanol; the hands, with gloves had to be cleaned with ethanol;

3c. Step-wise scaling up would not result in the cell getting a “culture-shock”. In this way, it is also possible to monitor the optimum growing conditions rather than in a large scale medium. If in a large scale medium, it is very difficult to measure the culture parameters. Also, there are considerations to be decided before upscale such as Stir rate, Temp of media, Factors affecting yield of prod.

Nic & Fil

Hey people! Today BPT practical was great because it was a very short one! J We transferred several colonies of GFP(Green Fluorescent Protein) transformed E.coli from a fresh LB/Amp/Ara plate to the flask containing 100ml LB medium with ampicillin. This will be used to inoculate the fermentor for scale – up fermentation.

For a clearer understanding, please look at the video below.




I know what is running through your mind... "What is GFP" and "how it is incorporated in E.coli" rite? With Meena and Shu Fung here why worry?! We’ll tell you the answers!:P The green fluorescent protein (GFP) is bascially a protein comprising of 238 amino acids which originated from the jellyfish Aequorea victoria. The structural shape of the GFP is a beta barrel, a cylindrical anti-parallel beta sheet comprising of 6 alpha helices and 11 beta strands interconnected together by loops. By now... all of you should know what those complicated words mean…..and thanks to Mr. Michael for that! But if you are still blur... Here is a picture to make your life easy!


(Source: A website that most of us cope introduction for reports.... WIKIPEDIA!)


What’s so good about GFP that it is used in research is its ability to fluoresce green when exposed under UV light. Cool right?! Due to the usefulness of GFP, it is used as an indicator and as a bioreporter by the scientists.



Now... to the part where how GFP is incorporated in E.coli...

The GFP gene is incorporated in E.coli by first inserting GFP gene into the plasmid DNA known as the pGLO. The pGLO protein also contains antibiotic resistance gene in it which will only allow transformed bacteria to grow in an agar containing antibiotic like ampicillin. Subsequently, transformation solution is used to heat shock the pGLO plasmid into the E.coli cell. Heat shock is often done to increase the permeability of the cell membrane and allow uptake of the pGLO plasmid into the cell. Following that, the cells are allowed to grow such that it will eventually express the GFP gene and form protein. (It’s sooooooo MBT!)


We are not finished yet!! We found a cool picture on GFP on the net….and we want to share it with you!... Imagine if a GFP gene is engineered…. and there are mutants obtained from it... When u streak it on a plate... How do you think the outcome will look like?
Can you picture it???



This is how it looks like!!! Bet you didn’t imagine this even in your wildest imagination!!:P It is a San Diego beach scene drawn with an eight color palette of bacterial colonies expressing fluorescent proteins derived from GFP.

That is all from us!

Till the next time we doodle...

Love. [Ishy & Shu Fung]

Whatsssuppp Peepers, woops I mean Peeps!!

As usual, we had bioprocess today and Immanuel and I are in charge of the Experiment 3…Which is the usual scientific inoculation, fermentation and Monitoring...Wait a min, no class monitor how to do monitoring?? LOL.Anyway, what happened today was pretty simple and procedural (Since when science is not procedural?) Basically, the laboratory technician or lab tech for short (why not VTEC?) transferred the seed culture (the culture dun have seed, but got the glowing E.coli cells) into the fermenter using the peristaltic pump. By doing so, what this VTEC woops I mean lab tech is doing is to allow the E.coli cells to grow at a larger scale (1 L is like large scale...-_______- "" ). After that, the lab tech then help us to collect the culture at T=0 as it is to be used as a control (or blank)...See, scientist always like to use symbols like T=1, [S] = 20,etc to make people feel that it is so deep, actually is much simpler. Okay, here comes the boring part, wait is boring or bo ring coz bo ring mean no ring in hokkien…

Er..Right. Basically what we have to do next is to assign our team members (2B), not 3A,3B or 6A,6B, this one not multi-storey car park! After assigning the team members, a pair of peeps will then collect the culture at every 1 hour interval for OD reading…So we are all good students such that the lab tech helped us to collect the culture at the last collection time which is 6pm. Actually right, no I mean left, the lab tech was very, ultra, super, ultimately nice to do it for us volunteeringly.

Procedure, Procedures, Procedures

1. Loosen the clamp on the input tube followed by pulling the plunger (backside) of the syringe to draw out the culture from the fermenter. The culture will then flow to the sampling test tube located on the side of the fermenter. Juz freaking push the plunger until the end.
2. After that, remember to tighten the irritating clamp back on the input.
3. Continuing on with the so boring procedure, loosen the clamp on the output tube. Next, pull out the syringe from the exhaust (How big is the size of the exhaust? Got 3" or 4" piping or not?) and air filter and pull the backside of the syringe.
4. Next, cap the syringe back to the exhaust air filter (Change Air Filter for more Horse Power!!!!) and push the backside.
5. The medium will flow to the collecting test tube and tighten the sickening clamp once and for all.

*Note that steps 3. and 5. is crucial. Failure to do so will result in eXpLoSiOn! Juz kidding…This is because if no tightening is done, the medium in the sampling will flow back to the fermenter, which might result in contamination.

*Step 2. is also required to ensure that the air is pushed back into the fermenter. Contamination will not occur in this case as there is an air filter (make sure that air filter got performance!) while pushing air back into the fermenter.

The sooo INTERESTING answers to questions are next!

1. For pH (Not referring to Philippines), the pH of the medium is detected by pH probe in the fermenter. Maintaining optimum (So Optimus Prime will come out), pH is controlled by the base and acid pumps on the automated machine. So if the pH is too acidic or too low, the base pump is activated by the machine and NaOH (Sodium Hydroxide) is added until the desired pH is reached. If the pH is too basic or too high, the acid pumps is activated and H₂SO₄ (Sulphuric acid) is added until desired pH is reached.
2. For temperature, in the batch fermentation process, the cooling jet (how bout jet plane? Not funny right?) will be activated if the temperature of the fermenter is too high.
3. For dissolved oxygen (how about unsolved ones? Hahahaa), the dissolved oxygen probe will measure the amount of dissolved oxygen in the medium to check whether it is sufficient or not. If it is insufficient, air will be released into the medium via sparger.

After all of that, Imauto and I, woops I mean Immanuel and I went to do the OD readings of what we had collected. Below is the table of the readings! Too bad got table but no chair, cannot sit down... haha...

Sample	Hours	OD600	log (x/x0)
Control	0.0	0	-
1	1.0	0.152	0
2	2.0	0.189	0.095
3	3.0	0.789	0.715
4	4.0	1.152	0.880
5	5.0	1.570	1.014
6	6.0	1.800	1.073
7	7.0	1.940	1.106
8	8.0	1.980	1.115
9	9.0	2.030	1.126
10	10.0	2.040	1.128

From the different samples we took, we determined the optical density (OD) of the sample using a machine called a spectrophotometer. This machine uses light absorbance to determine concentration of a solution using the formula using the equation A=Elc. Because terms E and l are constant, the equation can be phrased as A=c. The higher an OD reading is, the more light is being absorbed and thus more concentrated. 600 is the wavelength in nanometers used for this cell density. Why? Cos this amount of light is optimally absorbed by cells and not other contents such as waste and nutrients. Cool huh?

The readings were then log-ified to another term used for cell growth. A graph was then plotted to show how the bacteria has grown over the 9 hours. As you guys can see, the cells are growing in the log phase. The protein GFP is a primary metabolite (produced during growth). Ideally we should have harvested the protein before the cells starts dying (stationary phase!). But... we are not those kinds of people who stays in school. So we will harvest the protein tomorrow when the cells begin to die (death phase).

Immanuel and Ice Man (Shang-Feng ) Signing out... Till then, Ja ~ ne~

Hi bloggy, today was experiment 4 and what a slack day for us. We didn’t do anything. Our lab tech helped us do the final step which is to harvest the protein. He also showed us a history chart of all the parameters that were used in the fermenter.



Aren’t computers just dandy? They do the hard work for us. By setting the limits, the computer will control the optimal range of values for the cells to growth. For example, the green line represents the stirring speed. This line fluctuates soooo much as it tries to bring the amount of dissolved oxygen to optimal levels. The blue line also fluctuates together with the green line. All the parameters are kept at a certain range. Never does it goes beyond the range. Cheers to computers. Imagine what would happen if these things were not computerized? We’ll be camping in the lab measuring the pH/temperature/PO₂ of the fermenter and try to stabilize the parameters to an optimal range EVERY waking moment. How convenient computers have made our lives!

Well I better sign off here. Next Monday will be a long one. 3 hours of fun. Can’t wait for it! Peace out bloggy!

Immanuel

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 retardedcold 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!