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

~~~~~~~~~~

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"!

~~~~~~~~~

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 ------

Monday, November 12, 2007

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.

- Teck Hui and Wenyi

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.

- Thow

Brought to you by: MB0603 (Grp 3B)

11:50 PM