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.