*Fall in love with GFP like we did.*

Monday, December 3, 2007

Day Five

12:21:00 PM

Day 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:

Stage 1: Isolation

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).

Other pictures for the same process:

(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

This is to purify the GFP protein from unwanted protein that might have bound to the protein.

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.

There are two sections for Day 5:

You are Here: Part I (Procedures)

Part II (Results and Analysis)

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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