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Now that we have mastered incredible GFP technology, our next stop: global domination & world peace!
But first, we have to state our overall objectives of this practical: 1. To experience how the scale-up process works 2. To have better understanding of how the bioreactor works 3. To know how to use the bioreactor (1) What exactly is Green Fluorescent Protein? Who were the brilliant scientists that contributed to the discovery of this magnificent protein? To find out more, click on THE SECRETS BEHIND GFP. (2) We went through a lot together to try and produce functional GFPs in our darling, beloved Escherichia coli cells that we So browse through the links below for an intricate day-by-day analysis on what transpired during our experiments: (3) No one died during these five days. We swear! But through it all, we all had our own personal lessons to be learnt. Please check out INDIVIDUAL LESSONS to see what we have gained from this practical/project. (4) Our REFERENCES list, which contains various sources that we used to understand our experiment better. If you have time, check this out:
Welcome to Joy of Bioprocess!*This blog is best viewed under Internet Explorer. For some asinine reason beyond our understanding, programs such as Mozilla Firefox will screw up the entire format. So don't say no one warned ya!-----------------------------------------------------------------------------------------This web-blog is done by members of MB0606 Group A, which covers our experiences in cultivating a unique protein called Green Fluorescent Protein (GFP) in a strain of bacterial cells. Because we luuurve Bioprocess so much! :)Refer to the navigation links below to get around: sayang love a lot. My precioussssss...
MEMORIES THROUGHOUT THE PROJECT
Click on GROUP INTRODUCTION!
Friday, December 21, 2007
Please note that making funny faces intentionally is illegal in Singapore. It is a crime! These people above are wanted felonies! Please contact the authorities if you see any of them stalking a polytechnic near you :) Anyway, these are the group members who each contributed and made this project a success: - Sherry: our undisputed leader! Without her guidance, we'd be really lost. But it seems that Sherry's finger and nose has fused together for some reason in the picture above, so we sent her for plastic surgery last week. We have not heard from her since. - Hendro: Our resident BoyGenius. All hail Hendro! :) - Ziyi: Our second BoyGenius. Long live Ziyi! :) - Erine: ''Who wan play mahjong wit me arr??'' says THE MAHJONG QUEEN!!! - Amir: the guy who is obsessed with female blonde artists, namely Carrie Underwood, one of the blondes from that complicated drama series LOST, Dr Sara from Prison Break and Evangeline Lilly! Boooo! (~courtesy of Maya) - JJ: Jun Jie likes Japanese girls. This is why we always carry one along in case we want to blackmail him to do something for us. - Haida: a.k.a OldManHunter! To restrain Haida, we carry around ten feet of raffia string just in case. This is for the good of the community, and for men older than 18, which is like, almost every guy in school. Run for your lives! - Maya: the blog genius who helped us link all the pages together! Long live McDreamy + McMaya! =) - Daniel: daniel the monkey lover...so glad that we are not doing a monkey project...if not, he will be too hyper and we will not be able to control him anymore! It's already tough controlling him from getting too hyper now... =X - Zhen Ann: happy boy is who he is..dunno why he's always smiling! Hmmm..hiding something ar, Zhen Ann?? :) But that's a good thing when we are all stressed up with this project!! - Yong Wen: the silent killer...who can just scare you to your wits by just being silent! Thus protecting us from evil forces.... Hmmm.. I think that's all.
And not only that... after the practical, we still had to design a blog by ourselves. So we worked hard...right until the last minute on Sunday!! Here are some pics of us going crazy: Haida never do work ar! Hendro busy at work...dun kacau! But suddenly...when the clock strikes twelve...something unexpected happened... DEATH BY BIOPROCESS!!! Rest in piece, Hendro. Sherry has been killed by the Ju-On curse! Haida passes away peacefully while hugging her laptop. Hmm...dunno why her corpse still laughing leh. Amir dies peacefully in Carrie Underwood's arms (in his dreams!) Poor Ziyi has been crushed to death by his own mouse! JJ is buried under tons of bioprocess notes...the worst place to die! Mr Daniel Tan...so unglam! =) Ooops. Aaaand that's the end of it!!! XD
Even through all the hard work and effort we had to put in, there was still some fun to be had! Below is a compilation of some of our most memorable moments during this project... almost all have been photoshopped! No feelings were spared! Hehehehh.... Haida finally finds her life partner... YongWen's favourite pasttime! CENSORED: Deleted scene from Titanic... No wait... Haida has found ANOTHER soulmate... Never pay attention in class! Goh Ziyi!! What are you doing!! Zhen Ann is terrified of... THE MAHJONG QUEEN!!!!! :) Mr Daniel Tan would like to destroy NYP with a non-threatening bottle of chemicals. No handphones in lab class! =) Other pictures we took from the lab...
Our Learning Comments & Final Say: so after all that hard work, what did we take away from the experience? We interview each other to find out each of our two cents: HENDRO: To me, I learnt how to identify the different parts of fermentor and its function. And then there are many parameters needed to monitor for doing scale-up. Its not easy to scale up the process. ZHEN ANN: I had learnt alot from this projects. haha too much things i dont think i can finish writing all here. Firstly i learned how to carry out a fermentation process in lab scale. I had learned how to recognize the various parts of fermentor and how these different parts took part in the fermentation process. Besides, i also learned to isolate and harvest our precious green fluorescent products after the fermentation process. For the report part, this is the first time i learned the ways to create a blog together with my group. I can say that i sacrificed alot of my "first time" for this project ha. Together with my members, we had work very very very HARD to complete this very very very HARD task. Hope u can enjoy our hard works. And lastly, i LOVE my team members alot hahahaha YONGWEN: Learnt how to manage the fermentor, the different parts and its functions.... Learnt that blog is super irritating if dunno how to use t! Hahah! Niwae, communication is important in a group proj! Soorrriii... caught me slping in lab! ahhaa.. Had a bz nite!!! Lol ERINE: Okay. Generally i have to admit that i have had too much fun in playing and goofing around that i forgot what i have learnt! haha. ok ok seriously, i have learnt that there are many steps in fermentation process and scaling up and i will never forget the day that every member of the group have to take a sample every hour. we have to stay back in school till so late just to take a sample from the cells! ZzZzzZ -_________- ZIYI: Hahaha. Is this our chirstmas gift??? ok kidding. Anyway, from this practical we applied what he have learnt so far from the lectures into action. It provided us with hands on experience of how a biomanufacturing sector is like, and whether we're interested in this job sector next time. Career planning you know! ^(00)^ MAYA: Hmm...I learnt that a fermentation process is very tedious...there are a lot of things we need to be aware of before scaling up any product from lab scale, like nutrient requirements, because our cells need a lot of care. And the parts of a fermenter is very very complicated...lots of portions to know before doing scale-up! And on the blog, I find it a little difficult to manipulate the complicating HTML codes even though I blog reguarly. But it was worth it! DANIEL: Aye.. what a good quesion to ask huh.The things that I have learnt are that we must be careful in every step of the experiment as one wrong step may result in difference in the result and not give us the best results. The problem would be that we are not IT students. hahas. So its hard for us to do up a blog and al the stuff. hahas. we don't understand html codings. hahas. :D HAIDA: What we learnt... hmmm... since this practical is on bioreactor... of course it will be the bioreactor itself! Learnt different parts of the fermenter... its functions and how to use it. Besides that... I learnt that doing a blog MUST be REALLY PATIENT! If not... think will either end up in IMH or prison for being CRAZY! Hahaha! Niwae, learnt some new things on doing this blog! :D AMIR: wait! I thought this entire blog contains ALL OF OUR LEARNING POINTS?? so is this an exam qn? must make mind-map not? :) anyway, what I've learnt... is that E.coli likes to haunt us everywhere we go, even though we carry those little things in our stomach everyday... and that even though scaling up might sound easy in theory, like u add this, add that, like making rojak, it is actually very tough to do experimentally. Have to pay close attention to details and parameters like oxygen requirements otherwise everything goes wrong! And you end up in prison like my friend Haida! Right, Haida??? And then break out with W.Miller right! =) JJ: Hmmm o.o i've learnt... well what have i learnt? o.o probably learnt that scaling up isn't as simple as getting a bigger container and chucking everything in XD and even though the actual experiment itself isn'y very confusing, the making of the blog, the compilation of the data and analysis is really tedious and complicated. The biggest problem encounter was probably the meeting dates and timings.. whatelse...? hmmm i think i need to work on my discipline too.. *sighs* SHERRY: Hmm..what have i learnt? what have i learnt? nothing? haha...kidding...i've learnt that scaling up is a tedious and somehow a troublesome process! many parameters/information are required for the scaling up process. And in the process of doing this project, we've met with many problems along the way...like making a blog when we have no great knowledge on doing one! but we overcome it wif everyone's help and contributions! with thanks to all my group members... :) In conclusion, our learning points in general are:- - Learned how to scale up the cells. - Understand parts of fermentor and its function - Parameters involved in monitoring scale up. That's all folks! Thanks for taking the time to visit us!
A day after the practical blog was finally completed....
Day 5, Part II Before doing any distruption cell method, we must do centrifugation to isolate the E.coli cells. The centrifugation would use gravitational force to pull down all suspended cell into the pellet at the end of the tube. Since the GFP protein is an intracellular product, it is necessary to lyse and extract the product from the bacteria cells. These are some of the disruption methods that were performed on the bacteria cells: (1) The first method used to lyse the E.coli cell is through the use of enzyme. The lysozyme is the enzyme used to lyse E.coli cells. Before addition of lysozyme, the cell pellet was resuspended in TE buffer to provide good condition for lysozyme to work with. The addition of lysozyme would break open the cell wall and membrane, leading the release of all contents which were initially inside the cells. These contents would include our desired protein, which is GFP. (2) The second method that we used was freezing and thawing method. This method involves putting the tubes in liquid nitrogen (-120 degrees Celsius) to freeze the cells and putting the tubes into warm water to thaw the cells. These freezing and thawing add mechanical stress to the cell wall as the cell water content expands when it is frozen and contracts when it is thawed. (3) The last method was sonication. Sonication usually generates ultrasonic waves. These waves cause bubbles to implode violently and free radicals to form, thus disrupting the cell. Purification to get rid of other undesired products The technique that we used in this experiment was size exclusion chromatography. Size exclusion chromatography separates and purifies proteins base on the size or molecular weight of the proteins. Smaller proteins will be retained in the stationary phase or matrix which will get eluted out at a later time as compared to larger proteins that are retained in the mobile phase. Results Discussion for the isolation and purification of GFP in the form of chromatograms: Our comments on question 1: JJ: Sherry: Amir: Sherry: Ziyi: (All stare at him with that cold look…haha) YongWen: Our comments on question 2: Sherry: Daniel: Hendro: Yongwen: Amir: Haida: Ziyi: JJ: Maya: JJ: Hendro: (Erine suddenly enters the room) Erine: There are two sections for Day 5:
Other pictures for the same process: This is to purify the GFP protein from unwanted protein that might have bound to the protein. There are two sections for Day 5:
Our Beloved Group Members: 
*click on the above photo to see an enlarged and scarier version of our faces! Seacrest amiR, out!
Memories We Shared Part II
2:29:00 AMMemories We Shared Throughout the Project [cont'd]
In conclusion...
Thursday, December 20, 2007
Memories We Shared
7:42:00 PMMemories We Shared Throughout the Project
Poor Sherry has forgotten how to use the pipette!
Tuesday, December 4, 2007
Our Learning Comments
1:57:00 AM
Day Six
1:30:00 AMDay 6 
Monday, December 3, 2007
Day Five Part II
10:00:00 PM
Day 5 also includes the isolation of the desired product, our beloved GFP!
Fractions (at OD 476nm)
Blank 0.000
Tube 1 0.000
Tube 2 0.044
Tube 3 0.050
Tube 4 0.012
Tube 5 0.012
Tube 6 0.006
Tube 7 0.012
Tube 8 0.000 .jpg)
O.o where’s the blank?
Ya... The weird program did not let us change the labeling.. so in the end we did not put it -.-
Ya… the stupid program don’t let us change the axis labels! So cekik dara!
Should say we dunno how to use the program!!
Then, what to do now?
Discuss the thing? Frankly I don't see much to discuss about.. Its obvious that most of the GPF are eluted in fractions 2 and 3 .. Even a blind guy can see! ...
JJ:
okay... so maybe not... =.=
Amir:
Ok… back to the question, it’s clear that there’s no more GFP in the column after the 7th fraction cause fraction 8 has 0 absorbance like fraction 1.
Ziyi:
ok... so what now..?
Why the first one has 0 Absorbance?
JJ:
That was the first fraction we collected so the GFP haven’t pass through the column yet…
DANIIIEEEELL!! What do you have to say about this?!
How I know what I'm supposed to say... ask Hendro! Ask him!
Ar? Ehhh... Let me read the question first...
GFP is smaller than the 50,000 kD protein and we used size exclusion chromatography right?
Abo.
I think the protein with 50,000kD should be eluted before the fractions of GFP.
Hmm.. Ya, I think so. Cuz size exclusion lets the larger proteins elute first due to higher retention time in the mobile phase ma.
You are just reading off the lecture notes! XD
Wait... longer time in mobile phase... so?
Hmmm... longer time in the mobile phase means it gets eluted faster than the smaller proteins, which hets trapped in the "pores" or "tunnels" created by the matrix of the beads.
Erm.. so is that all? I can’t think of anything else to say... Everyone dismissed!
MAHJONG TIME!!! =)
Part I (Procedures)
You are Here: Part II (Results and Analysis)
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Day Five
12:21:00 PMDay 5, Part I
Experiment 4: Isolation and Purification of Product
Objectives
1. To understand the different methods of isolation of GFP product (Stage 1)
2. To carry out purification method - via size exclusion chromatography (Stage 2)
For Day 5, we intend to isolate and purify the GFP that is present in our cells. If we had done the previous steps correctly, then we should be able to see visible signs that GFP exists within our cells soo. But first, we had to centrifuge down the cells at 10,000rpm for five minutes: 
After centrifugation, we can see some GFP in the tubes under UV light. It's raining GFP! Hallelujah! We are so proud of our babies:
This is to isolate the GFP protein from the cells. There are three different methods that can be carried out to isolate the desired product. (a) Using enzymes
In this method, the pellet formed after centrifugation was resuspend in 500µl of TE buffer of pH 7.5 using a micropipettor. Lysozyme, a type of enzyme, was used to initiate the enzymatic digestion of the bacteria cell wall. The reaction of enzyme-bacteria lasted for 15 minutes.
(b) Freezing & Thawing
In this method, liquid nitrogen was used to freeze the contents while warm water was used for thawing. The freeze-thawing cycle was repeated for several times to complete the rupturing of the bacteria cell wall. This is because these actions add mechanical stress to the cell wall as the cell water content expand (when froze) and contract (when thawed). 
(c) Sonication
In this method, ultrasound waves causes the bacteria cell wall to breakdown under vibrational pressure. Since the process releases a lot of heat, it is important to carry out this process in ice. This is also to prevent proteins within the cells to be degraded.
Stage 2: Purification 1. Eight test tubes were labeled (from “1” to “8”) and a “blank” were placed in a rack.
2. The blank was filled with 2.0 ml of ammonium bicarbonate. Using this test tube as a guide, the rest of the test tubes were marked with a line at the 2.0 ml level.
3. The column was carefully drained into a waste beaker until the buffer is just even with the top of the gel bed.
4. Using a disposable glass pipette, the cell-free extract were transferred to the top of the gel bed by gently swirling the pipette around the inside edge of the column, just above the top of the packed matrix.
5. Prepared to take fractions by removing the waste beaker and placing a test tube under the stopcock. From this point on, the buffer (eluent) will be collected in test tubes. Each test tube will be filled to the 2.0 ml mark made in step 2 before moving on to the next tube. Each filled test tube is called a fraction.
6. Began taking fractions now. The stopcock was slowly opened and the sample was allowed to flow completely into the gel bed, and the eluting buffer was collected in the first test tube. The flow rate was adjusted to a 1 drop/2 sec interval.
7. 50 mM ammonium bicarbonate buffer was added carefully to the top of the column while taking fractions. A 2-3 cm “column” of buffer on top of the gel column was maintained to provide consistent flow of buffer through the chromatography matrix.
8. 2 ml fractions were continuously taken until the 8th tube is filled.
You are Here: Part I (Procedures)
Part II (Results and Analysis)
Back to Homepage
