Green Fluorescent Protein

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 =)

------ The Discovery of GFP ------

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. =)

------ Discovery of GFP - Osamu Shimomura ------

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

------ What is GFP? ------

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!

------ GFP Production Method ------

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! =)

------ The Team! ------

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 =)

------ The Fermentor! ------

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!

------ Parts of the Fermentor ------

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.

------ Our GFP Production Journal ------

Wednesday, November 7, 2007

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.

- Cheng Kong and Yi Ting

Brought to you by: MB0603 (Grp 3B)

11:47 PM