Bioprocess Report

2007-11-26T19:24:00.000+08:00

Day 5

Overall objective for this series of experiments
-To isolate GFP from E.coli using fermentation methods.

Goals for the day
- To isolate the bacteria containing GFP harvested from the fermentation process, via 3 methods.
- To purify the GFP isolated from the bacteria cells using Gel Permeation Chromatography.

Today is another long prac, estimated time of ending was 6pm. 3 to 6. What a long time.[TO ALL THE PEOPLE OUT THERE THAT WANTS TO JOIN MOLECULAR BIOTECHNOLOGY]. Think about it before u join. If not dont complain about the prac hours after u join this course.
Hope we dont get marked down just because we made this statement about MB. After most of us arrived in the lab at Q.604, Mr Ong started the briefing on the last experiment (OH YES FINALLY). He went though the procedures of the experiment, which is the different steps of isolation, purification and the analysis of the products.

After the briefing, we got down to do work(DUH!). We started with the isolation part. The isolation process started with separating the suspension into supernatant and pellets and into 2 tubes via centrifuge. When we went to various labs to use the Centrifuge machine, they are either in use or not suitable for bacteria cells, so we had to walk here and there to see if there are any centrifuge machine that we can use. Finally, we got to use a centrifuge machine and there we left our tubes to spin while we went to SLACK(well not all of us). After centrifuge was done, we took the 2 tubes and placed them at the UV machine to check whether the GFP is still there, and of course, its there. Haha. Aiyo, the spectrophotometer was an 'ANTIQUE' sia.. So old liao, is time to change a NEW one instead.. HeHes.. Below is the picture of our tubes with the GFP at the bottom:

After looking at the GFP, we had to lyse the cells to get the GFP, which is in the E.coli. Denash and Mark did the 1st part. They added TE buffer to resuspend the pellets and then lysozyme to initiate the enzymatic digestion of the cell wall. Halfway through shaking the tubes, denash started shaking n dancing sexily which got us all laughing like crazy!!! Dancing King Denash.. Shake ur BOTTY!!! Our Most Potential Dancer!!!

After the 1st part, we went on to do the 2nd part which is to freeze and thaw the cells. We get to use liquid nitrogen(looks like those wedding used de smoke) in this part to freeze the bacteria cells and then put the cells into HOT WATER (wow! sauna leh!) to thaw the cells. The process was repeated for 2 times which is a total of 3 cycles. This is to add mechanical stress to the cell walls of the bacteria cell as to help in cell lysis.

The last part of isolation was to use the sonication machine. This machine uses ULTRAWAVES to complete the lysis process. Before starting the machine, Mr Ong asked everyone to leave the room. But after awhile, wei hao went into the room and started his 'nonsense' about the sonication machine. Denash joined in the fun too after that and then eventually Jing Ye went in too. The guys was inside making a fool out of themselves while the girls were outside filming like nobody's business. All the girls very KIASI!!! They are afraid that if they go in ultrasound will cause them to be deaf or CANT CONCEIVE, according to huiyee's THEORY(nonsense theory actually) :D

After the cell lysis procedure, we centrifuged(AGAIN) the lysed cells to separate the GFP and the lysed products. So while the centrifuge machine was running, Jing Ye and Wei hao were playing their PSP again (Always play play play, aiyoyo!). This time we caught them 'eating snake'. Hahas, this time see how u two escape! After that, weihao & gang were fooling around with the whiteboard. Weihao was thin enough to go in between the boards.. hahaha.. he should eat more to fatten himself up!After the centrifuge was done, the tubes were decanted and put under the UV machine to screen for the GFP and also marked the end of the isolation process.

After the isolation, we had to isolate the GFP obtained from the cells. Gel Permeation Chromatography(so long time!! Cant the process run FASTER!) was used for the purification process. This method works using a column of polymer gel called the Sephadex G75 which has many many small holes, or rather pores in it. This separates the larger molecules from the smaller molecules thus the bigger molecules will reach the bottom of the column quicker and the smaller molecules will diffuse into the small pores of the gel and then reaching the bottom of the column slightly later. 8 fractions of sample were collected and they were put into curvettes for spectrophotometry. Wah sei, they still can be so happy sia.. Really know how to 'ku zhong zuo le', impressive!!!

After the spectrophotometry process, values were obtained and we had to plot a graph(graph again, sian) of OD at 476 nm against the fraction number. This MARKS the end of our BPT prac(YAY!! FINALLY!!).

We would like to tkank all the people who came to see our blog, and of course, not forgetting the lecturers and lab technicians which helped us to do our culture and fermenter throughout this prac. We hope to see all of you again (NOT in BPT prac) and thank you for all your attention . =)

2007-11-22T23:10:00.000+08:00

Day 4

Welcome to our 'channel' again! Today our job were simple compared to ytd. We did not even ~~need~~ to wear lab coat as the lab tech told is okay as we were not going to do anything.. Our culture is being transferred out to a new flask, it is no longer in the fermentor. We thought that our culture had went MISSING but later found out was a false alarm.. It just had been transferred out to another flask by the lab technician.. As the other grp's culture is still in the fermentor, he then demo on how to take out the culture from the fermentor.. The steps are similar as how we took out our sample on ytd..

After that, we went to the next lab to look at our history plot in the computer and a copy of it was given to us to keep.. The history plot showed the 4 parameters:
- Temp value
- Stirrer value
- pO2 value
- pH value
on how it is 'apply' to the culture.. We thought of taking the OD readings but the lab tech told us that we could only take it on Monday.. HURRAY! Tml no nid to go for bpt follow-up, finally can take a rest and continue on Monday.. This is really a GOOD planning as tml is near to weekend, we at least can take a rest and enjoy our weekends.. Hahas!!!

So do continue to read our entries on Monday as to know whether did our expt succeed or it fail.. Story to be continued again........

2007-11-21T23:41:00.000+08:00

Day 3

Goals:
- 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.

Today was a tedious day for us!!! We had to take sample out at every hr, so troublesome! And somemore we haf to meet at 8am in the EARLY MORNING, it is so tiring sia. The lucky thing is that we split the job.. For 10am and 11am, nina and huiyee went take the sample, at 12pm and 1pm is siya and jieying's turn to go. The guys in our grp will take the afternoon shift all the way till 6pm.. Applause for them!!! We took a video of Denash, who was the last person (beacuse he lives near school, that's why!), doing the sampling. We had to take at least 9 samples out for our OD readings tml so tml there will be more postings up.. Rmb to stay tune to our 'channel'! Well, what a tiring day it was.. Story to be continued.....

FINALLY....

See, Jing ye was so enthusiastic about going home!!!

2007-11-21T22:45:00.000+08:00

Day 2

Tuesday 20/11/2007

GOALS OF THE DAY!!!
- To describe steps to prepare a bioreactor
- To prepare the media for seed culture and scale-up reactor
- To prepare seed culture for scale-up fermentation

Today's prac was very short (GREAT!!). It took only 15 mins to finish. Before everyone arrived for the prac, jing ye and weihao was playing PSP (playing AGAIN.. Hahas!) while waiting for the rest to come. After all the people came, all we had to do was to see whether the E.coli was growing properly with the GFP. After looking at the E.coli grew fine, we went on ahead to take the colonies to put them into the shake flask to culture them into our seed-culture. Jing ye (volunteer himself de) did the innoculation of seed-culture and then the shake flask was taken to incubate for the innoculation into the fermenter tmr.. So that marked the end of today's prac... We can have our longer break then.. HURRAY!!!
Story to be continued....

2007-11-19T20:17:00.001+08:00

Day 1

Goals:
- To familiarize with the parts and components of microbial and mammalian bioreactors
- To introduce the basic operation procedure of a bioreactor

Today is our 1st bpt prac. Our lab is on the 6th floor instead of the usual ~~7th floor~~. We went early to the prac which Mr Ong was ~~not~~ there yet. Once Mr Ong came, he started briefing us on what to do and separate us in term of grpings, we are grp 5B.

Our 1st task was to prepare the medium. We carried all the apparatus needed over to the other lab and carried our expt there. We need to weight out 50g of LB and top it up with 2L of dH2O. Due to the size of the weighing boat too small, we could only weighed out half-half. DENASH was the one who dissolved the LB in the dH2O. It was a slow process, so Denash had to do it slowly and the smell is 'TERRIBLE', it smell just like TOILET.

While Denash was 'hardworking' dissolving, other members were doing something else. Huiyee was singing non-stop that we are AFRAID that the glasses in the lab will be BROKEN by her 'LOVELY' voice...Hahas! She cant stop singing for a single second de.. OMG!! The glasses did not break as we STOP her from singing in order to save the glasses (if not NO $$$ to pay back sia)...Hehes! So she took out her camera and started taking photos of us and things in the lab (her always ACTION!) so that we have photos to BLOG!!!

Finally, the LB medium was done. Denash's effort was paid. Huiyee then carried the medium back to the original lab but her way of carrying looks weird, she looked like she was carrying ash of human.. Hahas! so funny sia.. Back to the lab, we will analyzing the different parts of the bioreactor. The bioreactor used was a 2L model, it looks really like a 'transformer' has a lot of wires and tubes attached. Luckily, this is only a small 'TRANSFORMER'!

After Mr Ong go through the different parts of the bioreactor, lab teach Mr Ming Chuan then demo on how to operate the reactor. We were given the chance to 'play' with the reactor, Jing ye was the 1st to try on the reactor.. After that, Mr Ming Chuan wanted a grp leader from our grp to demo on how to operate and Weihao was kana named our so-called grp leader as his surname start with 'CHAN'!

Last part before we end our prac, we need to do a streak plate and the volunteer is HUIYEE. She was kana sabo by us. So while she was busy streaking the plate, we then helped to take down her 'beautiful' photos (actually NOT that beautiful lah.. Huiyee will sure KILL nina liao this time). After that, we could return to our home-sweet-home(released) then.. HURRAY! Releasing eariler!! Can GO HOME to rest liao.. Hope the rest of follow ups will also be lidat... =)

For now, some CANDID SHOTS!

Do stay tune to our tml's exciting and funny experience! Well, shall end den.. <".">