*Fall in love with GFP like we did.*

Monday, December 3, 2007

Day Five Part II

10:00:00 PM

Day 5, Part II

Day 5 also includes the isolation of the desired product, our beloved GFP!

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

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

Discussion for the isolation and purification of GFP in the form of chromatograms:

The results for the isolation and purification of GFP in the form of chromatograms were measured by using spectrophotometry at the absorbance of 476 nm.
GFP absorbs and gives out its usual fluorescence strongly at this wavelength and thus higher absorbance readings will reflect higher amounts of GFP in the fractions.
From the graph, we can see that the absorbance increased and then decreased down the fractions.
The initial low absorbance value was due to the retention of the GFP in the stationary phase. The larger molecules in the solution sample eluted first instead of the elution of GFP (smaller size).
At this time, fraction did not contain GFP. However, as the fraction number increased, more GFP was being eluted due to the diffusion through the resins.
Then, the amount GFP decreased again, as there was lesser amount of GFP left in the column.

Our comments on question 1:

JJ:
O.o where’s the blank?

Sherry:
Ya... The weird program did not let us change the labeling.. so in the end we did not put it -.-

Amir:
Ya… the stupid program don’t let us change the axis labels! So cekik dara!

Sherry:
Should say we dunno how to use the program!!

Ziyi:
Then, what to do now?

JJ:
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..?

(All stare at him with that cold look…haha)

YongWen:
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…

Our comments on question 2:

Sherry:
DANIIIEEEELL!! What do you have to say about this?!

Daniel:
How I know what I'm supposed to say... ask Hendro! Ask him!

Hendro:
Ar? Ehhh... Let me read the question first...

Yongwen:
GFP is smaller than the 50,000 kD protein and we used size exclusion chromatography right?

Amir:
Abo.

Haida:
I think the protein with 50,000kD should be eluted before the fractions of GFP.

Ziyi:
Hmm.. Ya, I think so. Cuz size exclusion lets the larger proteins elute first due to higher retention time in the mobile phase ma.

JJ:
You are just reading off the lecture notes! XD

Maya:
Wait... longer time in mobile phase... so?

JJ:
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.

Hendro:
Erm.. so is that all? I can’t think of anything else to say... Everyone dismissed!

(Erine suddenly enters the room)

Erine:
MAHJONG TIME!!! =)

There are two sections for Day 5:

Part I (Procedures)

You are Here: Part II (Results and Analysis)

Back to Homepage

Day 1, Experiment 1: Familiarization of bioreactor and equipments
Day 1, Experiment 2: Equipment and media preparation
Day 2: Inoculation, fermentation and monitoring
Day 3: Sampling and monitoring of cell growth
Day 4: Harvesting of the cells