Bioprocess Technology Practical: Day 5 - Judgement Day...! Isolation and Purification of Product

Stage 1

Isolation

To obtain Green Fluorescent Protein (GFP), firstly we need to lyse the bacteria cells to release the protein.

10mL of culture broth is collected and then centrifuged at 10,000rpm for 5 minutes to obtain the cells, which forms the pellet at the bottom of the tube. We observed the cells in the tube under UV light to ensure the product we want is in the pellet.

3 methods to disrupt the cells:

Use of enzymes

Freezing and thawing

Sonication

1. Use of Enzymes

The pellet is resuspended in 500 μL of TE buffer of pH 7.5. Then, 2 drops of lysozymes are added to the resuspended pellet. This will lead to the enzymatic digestion of the cell wall of the bacteria. The tube is left untouched for 15 minutes.

2. Freezing and Thawing

(Yea... here comes the fun part of the experiment! xD)

Firstly, the cells are frozen by placing the tube in liquid nitrogen (-196°C! wow that's cold! a sizzling sound can be heard...) Then, thaw it in warm water. Repeat these 2 steps 2 more times to break up the cell wall completely. Freezing and thawing expands and contracts the cell water content respectively.

3. Sonication

The cell walls of the cells imploded due to the vibrational pressure caused by ultrasonic waves. There are 4 cycles of 25 seconds of sonication, which 10 seconds rest in between. After that, centrifuge the cells at 10,000 rpm for 20 minutes to separate out the supernatant and the pellet. The pellet was then resuspended in 400 μL of TE buffer.

Now, the GFP is in the supernatant. We can double-check by viewing the tubes under UV light...

There! The fluorescent light confirmed that GFP is obtained in the supernatant! (:

Stage 2

Purification

Procedures

(HEY HEY, gel matrix: “cover me with buffer dudes/babes, don’t keep me dry”)

Eight test tubes were labeled and a blank was included to be placed in a rack.

The blank was filled with 2.0ml of ammonium bicarbonate.

The column was drained into a waste baker until the buffer is just even with the top of the gel bed.

Cell-free extract was transferred to the top of the gel bed using disposable dropper gently.

Buffer would be collected in test tubes to the 2.0 ml mark.

The sample was allowed to flow into the gel bed and flow rate was adjusted to be 1 drop/ 2sec interval.

50 mM ammonium bicarbonate buffer was continuously added to the top of the column while fractions taken.

2 ml fractions were taken until the 8th tube was filled.

Stage 3

Spectrophotometer

Spectrometer was used by a group of newbie researchers (WILSON, SADHA, ANDRE, JUNJIE, JARAMY) to record the absorbance readings at 476nm.

My Absorbance Readings:

Girls Absorbance Readings.