Hello. Welcome to Glowing Green Stuff.
This is a blog done by a group of Molecular Biotechnology students from Nanyang Polytechnic.
The aim is to provide visitors with a deeper understanding about Green Fluorescent Protein (GFP), and also to share our experiences during the production of GFP.
Note: For the best viewing experience, pls switch to full screen mode (Internet Explorer users plese press F11). Thank you and enjoy =)
As we all know, Mother Nature has created many glowing marvels throughout history; Stars glitter high up in the sky at night, and one can never forget the scenic view when fireflies take to the air.
As Man saw these, they were smitten by the allure of the glow from the fireflies.
In 1960s, scientists began to study these glow, and the concept of chemiluminescence soon evolved. In 1976, Richard Van Zandt filed the patent for the glowing lightstick!
Nice right? Lightsticks' glow looks really nice! ^^ - Jocelyn Chan
However, not all scientists studied the fluorescence in organisms via the chemical perspective; some did it from the biological way.
In 1960, about the same time when other scientists were looking at fireflies, a determined scientist began to look at the bioluminescence of a jellyfish called Aequorea victoria...
...Want to know who is that scientist? Click on "History of GFP" on the menu
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Wondering what exactly is GFP? Click on "What is GFP" =D
Want to know more about our group and our fermentor? Click on "The Team!"
Want to read about what went on during our GFP production? Click on "Our Journal"
Interested in our snapshots we took during our practicals, click on "Photos!!"
To come back to this page at any time, simply refresh the page. =)
In 1960, Osamu Shimomura was interested on the theory behind the bioluminescence of Aequorea Victoria, a crystal jellyfish.
It was in the lab at the basement of his home where the study began. He found that the glow comes from very small light producing organs found on the rings of the jellyfish. Hence, these rings were cut off from the jellyfishes, and squeezed to obtain the 'juice' that contained the GFP.
Ewwww! - Yi Ying
Throughout his study, he failed in many attempts to isolate the GFP. However, he was undeterred and his efforts eventually paid off -
Osamu Shimomura finally isolated his sample of GFP from Aequorea Victoria. The isolated GFP can be seen in the bottle held by him(above)
Over a million specimens were used! But, it was well worth it; his study led to the GFP revolution, where the protein was further studied and developed. It was eventually used as tracer molecules, and became a tool in understanding many aspects in cell and animal biology.
As such, Osamu Shimomura was called "The grandfather of the GFP revolution."
His story was published in the Volume 217 of Journal of Microscopy.
To read it: Download it here! (PDF Format)
Wondering what exactly is GFP? Click on "What is GFP" on the menu right now! =D
So what exactly is GFP? As mentioned earlier, it stands for Green Fluorescent Protein. It is 238 amino acids (26.9kDa) long and forms a 11-strand ‘beta-barrel' conformation. There is also a single alpha-helical strand which holds the chromophore that runs through the center of the protein molecule.
To let have a better idea of how it looks like, here’s the structure of the GFP:
The GFP Amino Acid Sequence:
MSKGEELFTGVVPVLVELDGDVNGQKFSVSGEGEGDATYGKLTLNFICT TGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FYKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNS HNVYIMGDKPKNGIKVNFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLP DNHYLSTQSALSKDPNEKRDHMILLEFVTAARITHGMDELYK
Because of its structure and its function, it is often called a “light in a can”.
The chromophore is the little red structure inside the greenish looking beta-barrel. This red structure is also commonly known as fluorophore (This is the reason why this protein will fluoresce!). This fluorophore is made up of three amino acids: Serine, Tyrosine and Glycine. Although this simple serine-tyrosinie-glycine motif is commonly found throughout nature, it does not generally result in fluorescent light.
In Aequorea victoria, GFP absorbs bioluminescent blue light from a photoprotein called Aequorin. This absorption of blue light, allows GFP to emit green fluorescent light.
So as you can see, GFP is as cool as lightstick! But if everyone wants to obtain this protein, Aequorea victoria could probably go extinct in no time (Not to mention about facing the wrath from a large group of animal activist)! However, with the advent of molecular biotechnology, production of GFP in the lab is possible! All we need is just a tiny bit of cells from Aequorea victoria =) It is quite a difficult task which requires the transformation of Escherichia coli cells.
Are you are curious about how it was produced in our lab?
Are you ready??..
..Here it is!
1. First, the cells from Aequorea Victoria were lysed to obtain their genomic DNA, which should contain a portion that encodes for GFP.
2. After the cells are lysed, the fragmented genomic DNA and pGLO plasmid vectors were cut using the same restriction enzyme.
3. In order to select the transformed cell at the later stage, pGLO vector contains two unique genes which codes for beta-lactamse and AraC.
4. Next, the fragments of genomic DNA were mixed with the plasmid vectors, in the hope that the particular gene sequence that encodes for
GFP will successfully ligate with a pGLO plasmid vector.
5. After the ligation step, the vectors were introduced into competent E. coli cells.
6. To isolate the transformed cells, the E. coli cells were grown on a Luria-Bertani agar plate with ampicilin and arabinose.
7. The colonies that grow on the plate should be bacteria cells which took up the plasmid vector (The vector contains beta-lactamase that can breakdown ampicilin).
8. In order to get E. coli cells that can produce GFP, the agar plate is placed under UV light to isolate fluorescing colonies.
(The arabinose that was added into the agar plate will “activate” the GFP gene.
9. Once the GFP-producing colonies were isolated, they are innoculated onto another agar plate (with same ingredients) to obtained pure cultures.
With the pure cultures of the transformed E. coli cells, large scale production of GFP is possible with the use of a fermentor! =D
That was the theory on how GFP could be produced. To look at what went on during our GFP Production in our lab, Click on "Our Journal"!
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To know more about us and our fermentor, Click on "The Team!" now! =)
We're currently Year 2 students from Nanyang Polytechnic! Molecular Biotechnology Rules! (Yup we're Molecular Biotechnology students)
From left:
(Front Row) Yi Ting, Yi Ying, Jocelyn, Chin Boon
(Middle Row) Cheng Kong, Affendy, Teck Hui, Choon Kiat, Thow (Xin Qiang)
(Back Row) Alan, Wenyi, Andrew =)
And here's the star of the whole show!! Our beloved mini-fermentor!
To view the location of the parts, click on the above picture!
The acid, base and antifoam!
The acid and base are used to adjust the pH of the culture
Acid: H2SO4
Base: NaOH
One of the baffles in the fermentor
Baffles are used to introduce random mixing, preventing one way mixing which is inadequate.
Condenser
It is used to condense water vapor to prevent excessive loss of water.
Control Panel: The brain of the fermentor!
It is used to adjust and maintain all parameters of the culture.
Cooling Jacket: The 'air-con' of the fermentor!
It is used to maintain optimum temperature of the media, it can warm or cool the culture to its desired condition.
Dissolved Oxygen Probe
It measures the amount of dissolved oxygen in the culture.
Exhaust Air filter
It is used to filter exhaust air to prevent air pollution. Although the exhaust air should be mostly carbon dioxide, the filter is generally present in culture fermentor to prevent any cases of toxic substance being released into the atmosphere.
Another important reason is to keep out contaminants from entering the fermentor via the exit.
Foam Probe
It detects the level of foaming in the culture
Impeller
The impeller stirs the culture
Inlet Air filter
It filters the air that is entering the fermentor
Motor
It is used to power the impeller
pH Probe
The pH probe is used to measure the pH of the culture in the fermentor
Pressure Gauge
It measures the pressure in the fermentor
Rotameter
It measures the flow rate of the air entering the fermentor
Sampling Tube
The sampling tube is used to draw samples out of the fermentor. Instead of drawing out the sample directly, this method minimizes chances of contamination
Sparger
The sparger introduces sterile air into the mixture
Temperature Probe
Is is used to measure the temperature of the media in the fermentor
The level probe (which is not used in this practical) is generally used to measure and maintain the level of the culture.
Final Words
This marks the end of a good week. That whole week, we learnt quite a bit of stuff on small scale lab production of desired protein. Now.. Its time for reflection!
Learning Points
Learning points of fermentation:
We get to know different part of the fermentor and its uses.
We learned the procedure to carry out scale-up fermentation.
Cells need to be at certain cell density before adding into the fermentor in order to reduce lag phase (cells get lonely too!!!)
Controlling of parameters during the fermentation process is crucial as any slight change may be disastrous to the cells
Day 6
IT WAS THE LAST DAY OF BIOPROCESS PRACTICAL!! HOHO!
Overall, the whole one week of bioprocess practical was fun even though we were only exposed to one tiny little fermentor and our cells didn’t grow as expected!!! Sianzz...but I guess we do learn quite a bit as this whole module teaches new stuffs…more of industrial basis and not the usual laboratory work…hahaha! Alright! Roughly, what we did that day was the isolation and purification of our product which is GFP! Green fluorescent protein! Something that glows …in green of course…
Isolation time!!! ^^
Okay! Serious time! Some brief introduction of GFP, it is an intracellular product. This means it is produced inside the cell. Therefore, to take out this cute little protein, we have to lyses the cell. =( There are three methods in cell disruption.
The bacteria cells are first centrifuged at 10,000 rpm for 5 minutes. Centrifugation separates things according to their density. Thus, the heavier object will be found at the bottom of the tube. Cool huh!!! =P Since, the cells are denser, they are found at the bottom of the tube which is called a pellet. The broth which the cells grow in will form the supernatant. This supernatant is then poured into another tube after centrifugation. The pellet is then observed under UV light!!!! Bloody dangerous NOT…got protection la…what are you thinking.
Because its going to be dangerous, better start looking at the procedure one more time before we all die from being blur.. Wahaha!
METHOD 1 – Using of Enzymes
The pellet is resuspended in 500µl TE buffer of pH 7.5. (Who don’t know what is pH…I make sure I kill you…ahem.) Then, two drops of lysozyme is added. These will initiate the enzymatic digestion of the bacteria cell wall. ahhhh!! The cells are dying!!! So ke lian = poor thing (Sounds so like YiYing -.-“)
METHOD 2 – Freezing and Thawing
The tube was placed into liquid nitrogen! Amateurs out there! Ever played with liquid nitrogen? No!? Okay…basically the tube is frozen and thawed in warm water; this cycle is repeated twice to rupture the bacteria cell wall. (YiYing keep quiet!) The freezing and thawing method add mechanical stress to the cell wall as the cell water content expands and contracts. What a harsh reality…tsktsk.
METHOD 3 – Sonication
This is a dangerous procedure if you stay inside the room too long…may go deaf? Hahaa! The cell is disrupted by using of ultrasonic waves that cause the bacteria cell wall to implode under vibrational pressure. This is repeated four times with 25 seconds sonication and 10 seconds rest. But this procedure is not interesting …and practically, you end up standing there and counting…boring! So be smart! Look and the person counting from outside. Right not YiYing! Wahaha!
After all 3 methods are done; the contents are spun through centrifugation. (20 minutes at 10,000 rpm) The GFP is found in the supernatant.
Glowing supernatant
PURIFICATION!
The GFP is purified using gel permeation! (Size exclusion chromatography) if more information u can go read up at Wikipedia and check okay...don't bug us…wahaha! Basically inside contains a lot these polymer gel resins and these resins contain very tiny pores where molecules can diffuse in. Therefore, larger molecules will come out first then smaller ones. This achieves separation of different molecules by size.
Size exclusion chromatography
After purifying, some samples are taken from the tube and added to the respective cuvettes…to go into spectrophotometer at absorbance 476nm! This is a wavelength where GFP strongly absorbs and gives off its fluorescence. You would sure hope that you were here! The green colour was fascinating…just something you wouldn’t get to see in your normal life… (Not saying scientists are abnormal la…ahem! Only one person abnormal here…YiY***)
Different fraction was placed into different cuvette
Sorry for being detailed …no choice hahaha! This blog is assessed okay…it carries marks! So whoever visited this blog and read it… leave a comment and thank you for your participation. WE of course have a prize for you!! ooohhh yes!! We do!! ^^ the prize is LIOW YIYING!! (She always complaint no one wants her and keep saying she pretty, so you know =P =P) YiYing for this project sacrifice a bit la…don’t get angry =X EVERYONE SAY THANK U!
THANK U!!!
(EVERYONE)
Disclaimer: We are not against YiYing. We are totally peaceful with her. Just that she…asked for it. =X so WE being nice…ya. You wouldn’t wanna know =] hahaha!!!!!!
Answer to Questions
1. Chromatogram Analysis
Green florescent protein (GFP) absorbs and strongly florescence when exposed to light of wavelength 476nm (nanometers). Therefore, an increase in the absorbance value would indicate that the GFP has exited the chromatography column. From the graph, it can be seen that the highest amount of GFP comes from fraction 2.
2. A protein with a molecular weight of 50,000 kD would elute into a fraction before GFP. This is because of the larger molecules from the solution will have difficulty entering the pores due to its size. Therefore, the larger molecule will pass through the gel easily and elute faster. The smaller molecules will enter the pores and get stuck inside the gel hence more time is needed to flow down. Thus, the smaller molecules will have a longer retention time inside the column.
Graph Analysis
Observations from the graph:
Graph measures 4 different aspects of the fermentor during the fermentation process: Temperature, pH, % of dissolved oxygen in the media and the impeller speed.
- Temperature remained constant throughout fermentation process: ~ 32˚ C
- pH: pH7.45 remained constant until 18hr 45 min and it started to increase to 7.55 where it is left to maintain constant for the rest of the fermentation process as it is the optimal temperature for cell growth.
- Dissolved oxygen: Maintained at 110% saturation before dropping and fluctuating between 10% - 30% .At around 32 hrs, steady increase until 80% saturation.
- Impeller speed: Remains constant unless the amount of dissolved oxygen starts to decline. Impeller speed will increase therefore more oxygen will be dissolved. It will return to normal speed when dissolved oxygen levels start to increase again.
From the graph, it is seen that Impeller speeds and amount of dissolved oxygen fluctuated the most during the 15th – 34th hour of the fermentation. This sudden decrease in dissolved oxygen levels indicate that the cells are taking in oxygen for biological reactions while the increase in impeller speeds show that the fermentor system is trying to increase the amount of oxygen dissolved in the media by stirring more. This period of high activity is most likely to be the log (exponential) growth phase of the bacteria cells. Before the log phase (0 -15hrs), there is no activity. This is the lag phase, the time required for the bacteria cells to adapt to their environment before replicating. The decrease in activity at the end of the 34th hour is most likely to be the cells entering the stationary phase when the cells stop actively replicating.
Day 5
Okay.. Its me again cause i simply love to blog! Today we went back to lab to obtain our graph. Hmm, from the graph it was quite apparent that the fermentor was shut down only this morning at 8.34am. I heard from someone that TSO said he would come back during Deepavali to switch off the fermentor and harvest the cells. Thinking about it.. who would come back to school on a public holiday anyway?? Haha!!
So here's the graph..
Hmm.. The culture probably did not go into death phase, so its probably okay. Our Escherichia coli was 'sleeping' (in lag phase) for the first 13hrs since innoculation anyway.. Hope that more of our GFP is produced as the fermentor was kept running for an extra night! Hehehehehe!
So in short: Today, the fermentor was shut down. The graph was obtained and the cells were harvested and stored.
PURIFICATION ON MONDAY! Hope GFP can be isolated.. *Prayzzz*
Day 4
I just woke up on my bed.. Its Day 4 of the practical.. Oh my! Am i late for school?
Phew..
HAPPY DEEPAVALI!!
Harvesting would be carried out on Day 5 instead. Our graph should be obtained by then.
Time to go back to sleep.. =)
Day 3
Innoculation and Fermentation
Today is Day 3, There was a bad news earlier, it seems that the seed culture did not grow well. To solve this problem, a loopful of culture from the cryovial, as well as another loop from the agar plate was added into the seed culture.
Lets take a look at the video on what is going to happen today. =)
We are at the laboratory to observe how the TSO (Technical Support Officer) insert the 100ml of the seed culture into the fermentor. After the culture was added into the fermentor, a 10ml sample was taken every hour. This procedure was carried out for a total of 10 hours. Reading of the OD measurement was recorded for each sample.
Monitoring of Fermentor
The G clamp between the fermentor and the sampling tube was first loosened.
The plunger is drawn back to suck the sample from the fermentor into the sampling tube.
The plunger is pushed back, to purge the tube of liquid.
The G clamp between the fermentor and the sampling tube is tightened back.
The syringe is taken off.
The plunger is drawn back.
The syringe is connected back to the filter.
The G clamp between the sampling tube and outlet is loosened.
The plunger is pushed back. The sample of cell culture is obtained.
Tbe G-clamp is tightened back
Results - Absorbance of all the samples.
The log (x/x0) row is not filled in because of several reasons. The first reason is due to low seed culture. During the practical, the bacteria were transferred from agar plate to the seed culture. As such, the bacteria in the seed culture required more time to remain in lag-phase. This resulted in almost no change OD reading, hence making it impossible to calculate log (x/x0) and plot the graph. Even the log (x/x0) is plotted out; all the OD readings were beyond the range between 0.2 and 1. This means the values deviate from Beer- Lambert law. Even if the graph was plotted, it is highly unlikely to be accurate.
It is observed that the OD readings were all very close to zero. This suggested that the cells were in lag phase for the entire 10 hours.
If the cells were growing at exponential phase, a standard table and log (x/x0) graph should be similar as shown below:
Answer to question
1. pH, temperature and dissolved oxygen can affect the growth of cells and they will not produce the product of interest if they are under stress. Most of the cells grow well between pH 6.5 to 7.0. If the pH is to go beyond this range, cells will lose it viability.
Cells need dissolved oxygen but solubility of oxygen is very low; often it does not dissolve well in culture broth. Hence methods to introduce oxygen into the culture have to be used. On the other hand, high concentration of oxygen could become toxic to the cells due to high level of free radicals. However, situations involving too high oxygen concentrations rarely occur.
Temperature should be properly controlled because cells will die if temperature is above 40˚C. In addition, cells cannot survive temperature fluctuations of more than 2˚C. Therefore it is imperative that temperature should be properly controlled.
2. In a spectrophotometer, light is emitted and split out into a spectrum. The appropriate slit will let through specific wavelength of light and the filtered light is absorbed by the substances present in the sample. Lastly, the detector will detect the transmitted light that has passed through. The system will then convert the transmittance into an absorbance reading.
(Base on the fact that 100% Transmittance = 0% Absorbance)
600nm was used because it is the wavelength at which the cells absorb maximally.
3. GFP is a secondary metabolite. The product should be harvested in the idiophase (are produced during late growth phase and in stationary phase). Our GFP product was actually harvested during the stationary phase.
4. Some advantages of using computer control system is that the computer can automatically detect changes in pH, oxygen concentration, temperature and help to maintain these variable at a optimum level for the cells to grow. It helps to cut off manually work of constantly adjusting value of these variables.
With the aid of computer control, results of our experiment could be tabulated in a short time frame.
Computer control system also display data in a very organized way.
It can perform certain function such as plotting of history chart.
From the history plot, the cells are actually in their lag phase trying to adapt to the environment for the first 15 hours, therefore there is no change in pO2 concentration. There is a sudden decrease of the pO2 concentration because the cells are now in log phase which have to utilize more oxygen for metabolism. From that time onwards, there was a continually decrease and increase in pO2 concentration during the interval from 15.0 hr to 22.5 hr. This show the effectiveness of the control system as when the pO2concentration is low which could lead to cell death, the concentration of pO2 was increased by the system. The system also ensures not to provide too high pO2 concentration as this may lead to free radical and kill the cell. Therefore the system help to ensure the pO2 concentration provided is not too low and high.
Another variable which can be use to show the effectiveness of the computer control is temperature. Work done by the fermentor and biochemical reaction of cells result in the production of heat which lead to increase in temperature. From the history plot, it can be seen that the temperature value is always increasing and decreasing. This is because when there is a rise in temperature, the control system helps to maintain it at a optimum temperature range for the cells to grow. Hence, this show effectiveness of the control system.
Stirring speed has a direct influence on oxygen concentration. Stirring help to generate oxygen but as say before, high amount of oxygen is harmful to the cells. During the first 15 hours, stirring speed remains constant. For the next 7.5 hours, there is a increase and decrease in the stirring speed because oxygen had to be supply to the cell for growth or else cells will die. If the oxygen concentration is too high, the control system will decrease the oxygen concentration, This show the effectiveness of the computer control system of providing the right concentration for the survival of the cells.
Day 2
The seed culture preparation was done on Day 2, Tuesday. We were scheduled to meet at 11a.m. to conduct the colony selection for our culture and to inoculate the colony into a LB/Amp media flask. The flask was then placed into a shaking incubator at 32ºC for 24 hours.
Before the preparation of Step-up culture, our streak plate from Day 1 was obtained.
Preparation of Step-up culture
Before we begin let us take a look at the procedures!
The preparation of the seed culture was done in a UV hood aseptically. Gloves are worn to prevent any contamination. Several colonies of pGLO transformed E.coli were obtained using a inoculating loop.
The colonies were transferred to the flask containing 100ml LB medium with ampicillin. Then the flask was shaked.
Dang! We were told to meet at 8 a.m. tomorrow! Sigh… Life of a MB student..
Day 1
Finally is the day we are going to start on our bioprocess practical! We were so enthusiastic about the practical that we went to class early. While waiting for our lecturer Mr ONG CHEE MING to enter the class, we put on our lab coats and tied up our hair to look neat and smart! When he opens the door of the laboratory our hearts start pumping as one. We started off by taking attendance and it is time for us to shout “HERE!”
Media preparation
We went over to another room to make our Luria-Bertani media.
But before we begin, let us take a look at the procedure once more!
Mr ONG was giving us instruction (:
Our Materials!
Choon Kiat weighing out 50g of Luria-Bertani powder.
Teck Hui pouring Milli-Q water into the glass bottle.
Cheng Kong putting in the Luria-Bertani powder.
Spinning, spinning, spinning~
And our 'chicken essence' is done! (we think that LB broth smells like chicken essence hehe)
Seed preparation
We were fascinated about producing green fluorescence protein within E.coli. Hands were swap before entering hood as we do not want to introduce any other bacteria.
Obtaining Inoculum from cryovial (Teck Hui)
Plate streaking (Teck Hui)
It was incubated overnight.
End of lesson ^-^
We look forward for the practical class tomorrow (:
Answer to Questions
1a. The ingredients found in Luria-Bertani Medium are bacto-tryptone, yeast extract, NaCl, dH2O and pH. These ingredients share common properties, which promotes cell growth.
Bacto-tryptone is a source of protein for the growth of bacteria.
NaCl provide sodium ions, which are used for transport and osmotic balance.
Yeast extract contains glutamic acid, which is also known as glutamate and vitamins. They are essential for cellular metabolism. Glutamate contributes as an intermediate in processes such as glycolysis, gluconeogenesis and citric acid cycle. Vitamins are bio-molecules that act as catalysts and substrates in chemical reaction.
Water was added to suspense the solids and pH was adjusted to 7.5, which is optimal for cell growth.
1b. The purpose of adding ampicillin in the media is to act as a selective tool. Ampicillin has an amino group and it helps to penetrate the membrane of gram-positive and some of the gram-negative bacteria. It acts as competitive inhibitor of enzyme transpeptidase, which is used by gram-positive bacteria to make cell wall. Bacteria, without the cell wall, could not survive. Therefore, only bacteria with ampicillin resistance are able to grow on the media.
In this experiment, the plasmids inserted into the host cell, which code for green fluoresce protein also coupled with the gene bla (beta-lactamase enzyme), which allowed transformed cell to be ampicillin resistant. Therefore, only the transformed cells, which carry the gene that encodes for beta-lactamse enzyme, are able to grow on the media. Untransformed cell, which is not encoded with the gene bla, were inhibited. In this way, by adding ampicillin in the media, one could sieve out the untransformed cell.
Also by adding ampicillin, other bacteria or contaminant could be minimized as only ampicillin resistance bacteria are allowed to thrive in the media.
1c. Ampicillins are antibiotics, which are made of proteins. During autoclaving at high temperatures, it will cause ampicillin to denature which result in losing its functions.
2a. Calibration of pH probe is to adjust the indication of the pH probe so that it is within a specified accuracy of the standard.
2b. HCl will cause corrosion of the metal parts of the fermentor when it accidentally leaks out from the tubing which results in spoilage of fermentor.
2c. pO2 electrode is made out of Teflon membrane (permeable to oxygen) chamber with saturated KCl solution, platinum cathode and silver anode in it. It works by the removal of electron from silver to produce silver ions. The current (the flow of electron) will then flow to the platinum anode where the oxygen will be reduced. The amount of dissolved oxygen is proportionate to the current.
The term polarization means a fixed voltage is applied between the electrodes. This would ensure that the current is proportionate to the dissolve oxygen.
2d. Peristaltic pump is a positive displacement pump for pumping a variety of fluids. It made up of a flexible tube fitted in a circular pump casing. The rotor wills compresses the flexible tube which forces the fluid pump through the pump.
3a. Arabinose in the agar allow GFP gene in the insert to be “switch on”. It works by binding to the araC repressor and change it conformation so that it could no longer block transciption of GFP gene. RNA polymerase is then able to start transcription.
3b. Seed preparation was done in the fume hood, which has been UV for 15 minutes and swap with 70% ethanol. Hands were washed with soap; gloves were worn and were sprayed with 70% ethanol before entering the hood. All equipment was sprayed with 70% ethanol before entering the hood. Sterile inoculating loop was used to take a colony from the agar plate and placed into the conical flask containing media, which is essential for cell growth. The conical flask was autoclaved as well as the media before placing the colony in it.
3c. Transferring directly into the culture will result in longer lag phase within the fermentor as it takes a longer time for the bacteria to adapt to the surrounding before entering the log phase. Step-wise would helps to cut down longer time in lag phase.
The day before..
Ooh our practical would be starting tomorrow! Before we begin we shall look at our objectives and procedures! I wonder what would it be like..
Its going to be complicated.. but i'm sure its definitely not going to be like this:
Haha! Ok now let us take a look at the objective and procedures!
Main objectives and Procedures
To produce and extract the green fluorescent protein (GFP) using Escherichia coli, which involve techniques of media preparation, preparing seed culture, inoculation of seed culture into fermentor, sampling, and isolation and purification of product GFP.
Experiment No. 1: Familiarisation with the Bioreactor and its Operation
Objectives
To familiarise students with the parts and components of microbial and mammalian bioreactor
To introduce the basic operation procedure of a bioreactor
Procedures
Media preparation
1. 50g of LB of pre-mixed LB powder was weighed on the weighing boat
2. 1L of milli-Q water and the 50g of LB pre-mixed LB powder were poured into the glass bottle. It was then placed on the stirrer plate for mixing.
3. Milli-Q water was then used to top up to 2L.
4. It was left on the stirrer plate for mixing.
Bioreactor preparation
1. pH electrode was calibrated using standard buffer solution (pH 7 and pH 4 or 9 depending on the culture)
2. pH probe, pO2 probe and foam probe was installed into the top plate Height for the foam and level probe was adjusted if necessary.
3. The addition agent lines for acid, base and antifoam were connected. The levels in the storage bottle were checked.
4. Other accessories such as exhaust condensers, air inlet and exhaust filters and manual sampler unit was installed. The water jacket is filled with water.
5. All cables except the temperature probe was disconnected. Silicone tubing except for exhaust filter and female STT coupling of sampling unit was clamped. All filters and sockets were covered with aluminum foil. Culture flask was allowed to autoclave at 121° C for 20 minutes.
6. pO2 electrode was polarise for 6 hour. pO2 probe was calibrated by aerating with nitrogen
Addition lines were connected to peristaltic pumps
Seed preparation
1. pGLO transformed E.coli was retrieved from the -80°C freezer.
2. E.coli was streak on LB/Amp/Ara plate with inoculating loop.
3. The plate was incubated for 24 hours.
4. Single colony was obtained from LB/Amp/Ara plate and transferred into 100ml LB medium with ampicillin.
5. The flask was placed into the shaking incubator for 24 hours at 32°C.
Experiment No. 2: Equipment, Media and Seed Culture Preparation
Objectives
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
Procedures
Preparation of step up culture
1. LB/Amp/Ara plate was removed from the incubator and viewed under UV light to see the green fluorescence protein E.coli.
2. A single colony was selected from the LB/Amp/Ara streak plate and was inoculated to the culture flask.
3. Culture flask was placed in shaking incubator and incubated at 32°C for 24 hours.
Experiment No. 3: Inoculation, fermentation and monitoring
Objectives
To carry out scale-up fermentation process to increase the yield of desired protein product (Green fluorescent protein in this case)
To monitor cell growth and product formation through manual sampling and computer data logging
Procedures
1. When the medium broth had cooled to below 50ºC, ampicillin was added to a final concentration of 100ug/ml and arabinose was added to a final concentration of 0.2%.
2. The temperature, pH, stirred speed, pO2 set point and airflow were recorded as 32۫C, pH 7.5, stirred speed min 10%, max 90%, 20% pO2 set point and air flow min 25% max 100% respectively.
3. Colonies and cryovial were inoculated from agar plate as there is no growth in the culture flask in day 2.
4. 100ml of seed culture was inoculated into the fermentor.
5. 10 ml of the blank sample was taken out before inoculation and 10ml of the culture was drawn out every one hour for 10 hours.
6. Fermentation broth was harvest and 10ml of the culture was transferred into the test tube.
7. OD reading of the ten cuvette sample that was taken out every hour was recorded.
Experiment No. 4: Isolation and Purification of Product
Objectives
To isolate GFP product
- Using enzyme
- Freezing and thawing
- Sonication
To purify GFP using Gel Permeation Chromatography
Procedures
Isolation
1. 10ml of culture broth in a tube was collected for the rest of the experiments.
2. The cells were centrifuged at 10, 000rpm for 5 minutes.
3. The supernatant was poured into another tube and compared with the pellet by observing them under the UV light to confirm the presence of product.
Method 1: Using Enzymes
1. The pellet was resuspended in 500µl of TE buffer of pH 7.5 using a micropipette till no visible clumps can be seen.
2. 2 drops of lysozyme was added into the resuspended cell pellet using a disposable 1ml plastic pipette.
3. The enzymes were allowed to act for 15 minutes.
Method 2: Freezing and Thawing
1. The tube was placed into liquid nitrogen until the contents are frozen. Then it was thawed in warm water.
2. This cycle of freezing and thawing was repeated for another 2 times to complete the rupturing of the bacteria cell wall.
Method 3: Sonication
1. The tube was placed into a beaker of ice. Sonication was done on ice for 4 cycles of 25 seconds with 10 seconds rest in between each sonication cycles.
2. The contents of the tube were spun in a centrifuge for 20 minutes at 10, 000 rpm after cell disruption.
3. The supernatant was poured into another tube separating it with the pellet.
4. The pellet was resuspended using 400µl of TE buffer.
Purification
1. Eight test tubes and a “blank” were labelled (from “1” to “8”) and placed in a rack.
2. The blank was filled with 2.0ml of ammomium bicarbonate. This test tube was used as a guide to mark the rest of the test tubes with a line at the 2.0ml mark level.
3. The column was carefully drained into a waste beaker until the buffer was just above the top of the gel bed.
4. The cell-free extract was transferred to the top of the gel bad using a disposable plastic pipette. The pipette was swirled around gently at the inside edge of the column, just above the packed matrix.
5. Fractions were prepared by removing the water beaker and placing a test tube under the stopcock. The buffer (eluant) was collected into these test tubes until the 2.0ml mark before moving on to the next tube.
6. Fractions were begun to be taken. The stopcock was opened slowly and allowed the sample to flow completely into the gel bed, collecting the eluting buffer into the first test tube.
7. 50mM ammonium bicarbonate buffer was added carefully onto the top of the column while taking fractions. A 2-3 cm “column” of buffer was maintained on top of the gel column. This is to provide consistent flow of buffer through the chromatography matrix.
8. 2ml fractions were continued to be taken till the 8th tube was filled.
Analysis
Spectrophotometry
1. Some samples were taken from the fractions to take absorbance readings using the spectrophotometer at 476nm. It is the wavelength where GFP strongly absorbs and gives out its usual fluorescence
2. A graph was plotted at OD476 against the fraction number.
Yup thats it!
Looking forward to tomorrow! =)
Haha! Welcome to our photo gallery! Just some random shots of us in action! Enjoy!
Group Huddle! Time to discuss on the approach..
Professor Affendy: You two, look here.. Pay attention..
YT and CK: Yes Professor! *Listens attentively*
Chin Boon in action! Pasting the autoclave tape onto the bottle cap.
NEWSFLASH: Thow has just signed a million dollar endorsement with LB-Broth Inc..
The situation is tense.. any distraction will ruin it..
A moment of chivalry: "Here miss, allow me to turn on the tap for you.."
Another tense, lip-biting moment..
Whirlpool, a new tool for hypnosis.
(Why is everyone so mesmerised??)
Andrew: Why is my darn pen not working?
Teck, 0603's most reliable man, drawing samples.
"Whatcha looking at huh?" - Our very own Choon Kiat as we got caught sneaking up on him =)
Our pride and joy - The GFP are in the cells!
Pippettes, test tubes, cuvettes - Clues tells you that we are in a lab
Deep in thought..
Affendy and Choon Kiat.
Thow, after earning his million dollars endorsing LB-Broth, got himself a babe!
Look how radiant he is! (He probably drinks some of 'em =P)
Green fluorescent protein - Wikipedia, the free encyclopedia
http://en.wikipedia.org/wiki/Green_fluorescent_protein
O. Shimomura
Journal of Microscopy, Volume 217, Issue 1, Page 3-15, Jan 2005
Van Zandt - United States Patent: 4064428
http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=4064428.PN.&OS=PN/4064428&RS=PN/4064428
Matt BenDaniel - Fireflies
http://starmatt.com/gallery/astro/fireflies.html
Glowing genes: A revolution in biotechnology
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1386127
History and What is GFP
Lim Choon Kiat
Andrew Oh
First Blog Entry
Andrew Oh
Day 1 Blog entry
Jocelyn Chan
Liow Yi Ying
Day 2 Blog entry
Alan Sim
Affendy
Day 3 Blog entry
Chia Cheng Kong
Chua Yiting
Day 4 and Day 5 Blog entry
Andrew Oh
Day 6 Blog entry
Teo Teck Hui
Ong Wen Yi
Learning Points(Final Entry)
Lim Choon Kiat
Graph Analysis
Thow Xin Qiang
Ong Chin Boon
Blog designs
Andrew Oh
Videos
Jocelyn Chan
Liow Yi Ying
Photographers
Teo Teck Hui
Jocelyn Chan
Thank you for visiting! ^^