Experiment 4 (Day 5)

Isolation and Purification of Product

Objectives

1. To isolate the desired product using enzymes, freezing & thawing and sonication to obtain an extract needed for purification.
2. To purify the product via gel permeation chromatography and separate impurities by their different sizes.
3. To analyze the wavelength at which the green fluorescent protein strongly absorbs and give out its usual absorbance by taking absorbance readings using a spectrophotometer set at 476nm.

In this experiment, the Green Fluorescent Protein (GFP) is isolated from the E. coli in the sample by cell disruption techniques (using enzymes, freezing and thawing and sonication) and then purified using gel permeation (a.k.a. size exclusion chromatography). 2 tubes of 10ml broth culture were made to under go the 3 cell disruptions and the purification stages.

Stage 1 – Isolation

The procedure for obtaining a cell pellet from the culture broth is shown below.

1) Firstly, we had to obtain 10ml of culture broth in two test-tube for the rest of the experiments.

2) In order to separate the cells from the liquid broth, we have to centrifuge the culture broth at 10,000 rpm for 5 minutes.

3) A pellet would be formed at the bottom of the tube as the cells are dense whereas the liquid broth is less dense and forms the supernatant.

4) The supernatant was then poured into another tube and both tubes were observed under UV light to confirm that the GFPs are inside the pellet.

The procedures for each of the 3 cell disruption methods are shown below.

Enzymatic Method

1) Firstly, the pellet was resuspended in 500µl of TE buffer of pH 7.5 with a micropipette until there were no visible clumps.

2) Next, 2 drops of lysozyme were added to the resuspended pellet whereby this will initiate the enzymatic digestion of the bacteria cell wall.

3) The enzyme was then allowed to act on the resuspended pellet for 15 minutes.

Freezing and Thawing Method

1) After 15 minutes, the tube was placed in liquid nitrogen until the contents are frozen.

2) Subsequently, the tube was thawed in warm water.

3) The cycle of freezing and thawing was repeated for another 2 times to complete the rupturing of the bacteria cell wall.

4) Freezing and thawing add mechanical stress to the cell wall as the cell water content expands (when frozen) and contracts (when thawed) causing it to rupture.

Sonication Method

1) The cell disruption is completed by the process of Sonication where ultrasonic waves (of higher frequency than sound) are used to implode the bacteria cell wall under the pressure of ultrasonic waves’ vibration.

2) Sonication is done on ice for 4 cycles of 25 seconds with 10 seconds rest in between Sonication cycles so as to prevent denaturation of the GFP protein.

3) Next, the contents of the tube were centrifuged after cell disruption for 20 minutes at 10,000 rpm.

4) The pellet was separated out of the supernatant as done previously during method 1.

5) The product (GFP) is now in the supernatant and pressence of GFP can be seen by observing the tubes under UV light.

Stage 2 – Purification

Purification to obtain the GFP protein was done on the supernatant obtained in the isolation steps using size exclusion chromatography. This method of chromatography uses a colum of polymer gel resins (Sephadex G75) which contains small pores as the stationary phase. When the extract flows through the column, smaller molecules interact with the pores of the resin and elutes slower. However, the larger molecules which are too large to interact with the pores will flow through the column faster.

The procedure below shows how this is done.

1) Firstly, eight test tubes were labeled from ‘1’ to ‘8’ and another test tube was labeled as ‘blank’.

2) The blank was then filled with 2.0ml of ammonium bicarbonate. Using this tube as a rough guide, the rest of the test-tubes were marked at the 2.0ml level.

3) The column was next allowed to drain into a waste beaker until the buffer is just above the top of the gel bed.

4) Subsequently, our cell free extract obtained from the isolation steps was transferred into the top of the gel bed by swirling around the inner edge of the column.

5) Next, the stopcock was opened and the sample was allowed to flow into the gel bed and the eluting buffer was collected in the first tube. While the eluant (buffer) flows into the tube, more eluting buffer (ammonium bicarbonate) was added to the top of the column to ensure that it does not run dry.

6) Once the sample has filled the tube to the 2cm mark, the tube was swapped with the next. This was done for the remaining tubes while ensuring that the gel bead does not run dry.

Note: The column was washed with 50ml of ammonium bicarbonate after the experiment so that it could be reused.

Stage 3 – Analysis

After the purification, the solutions from each of the 8 tubes and the blank were transferred to cuvettes and their absorbance readings were taken using a spectrophotometer set at 476nm. This was done for the other set of tubes obtained from the other set cell extract. 476nm is the wavelength at which GFP strongly absorbs light and gives off fluorescence.

The results showing the absorbance readings are shown below.

The graphs show the absorbance readings against their respective fractions of the 2 samples.

Click image to enlarge

Discussion

For discussion purposes, only sample number 2’s graph is used.

Since GFP absorbs light at the wavelength of 476nm, the absorbance obtained is indicative to the amount of GFP present. Hence from observing the absorbance readings and the graph fraction 2 has the highest amount of GFP followed by fraction 1 and fraction 3. Fractions 4 to 8 possess no GFP due to the 0.000 absorbance.

From the 1st fraction to the 2nd fraction, the absorbance increased from 0.385 to 2.745 and from the 2nd fraction to the 3rd fraction it then decreased to 0.054. Hence it can be deduced that the GFP probably had started eluting from the 1st fraction and stopped eluting at the 3rd fraction as a peak is observed spanning the from the 1st to the 3rd fraction as seen from the graph. The 1st fraction is lower in absorbance as the GFP protein requires sometime to elute through the column and would contain a larger amount of ammonium bicarbonate. The 2nd fraction is the highest as this is where most of the GFP is eluted from the column. The 3rd fraction has a low absorbance as almost all the GFP have eluted and only the remaining amounts are collected here.

Hence to obtain the purified GFP product, fractions 1 to 3 should be used and pooled.

Note: There are additional questions to help in better understanding of this experiment. The question and answers are located in the next post.