Objectives
- To be introduced to the various methods in which intracellular products can be isolated from the cells
- To be introduced to a method of purification of the products.
The absorbance was read at 600nm for the collected hourly samples. The results were shown below and a graph of log(x/x0) versus time (hours). OD readings were recorded. The log(x/x0) was calculated in the following method.
Log(x/x0) was calculated by log (A/A0) because
When t = 0,
A0 = Elc0 - (1)
When t = t,
A = Elc - (2)
(2) ÷ (1)
_A = _c_
A0 c0
Therefore, log (A/A0) = log (c/c0)
log (c/c0) = log(x/x0)
After which, a graph was plotted using the values obtained and calculated.
The graph is shown below.
The purple line represents temperature. It is observed from the history plot that the temperature of the reactor was maintained at approximately 32.5 degrees celsius. There were not much of fluctuations for the temperature. This was also the case for the dark blue line, representing pH. The green line, representing pO2 levels, dropped to a lower level initially. This is because the oxygen is being used up by the cells for growth. As such, the cell density increases as seen in the cell growth graph. This phase is known as the exponential phase. As the level of oxygen decreases, the stirrer speed, represented by the light blue line, increases to help in the distribution of oxygen to the cells. Normally, high levels of oxygen are not pumped in as high levels of oxygen are toxic. The levels of oxygen will fluctuate during secondary metabolism, as the cells will use up the oxygen and when levels of oxygen fall too low, more oxygen will be pumped in. The stirrer speed will adjust appropriately to suit the levels of oxygen. As such, it can be seen in the history plot that the green and light blue lines are fluctuating. The cells continue to grow at this stage as illustrated in the cell growth curve, where the cell density increases. The cell growth curve shown above is only from hours 0.0 to 10.0. As such, the stationary phase and the death phase of the cell growth is not observed.At around 22.5 hours of the history plot, the level of oxygen starts to increase. As lesser cells use up the oxygen, the oxygen level will increase. This shows that the cells have started to die. Since the level of oxygen is increasing, it is not necessary for higher stirring speed. Hence the stirrer speed falls back to the pre-determined value.
GFP was isolated and purified. The following were the steps conducted.
Stage 1
1. The cells were harvested from the bioreactor.
2. 10mL of media was collected and centrifuged at 10,000rpm for 5 minutes. This seperates the cells from the media, and GFP would be collected in the pellets as they are intracellular products.
3. The pellet of cells were kept and went through a total of 3 methods for isolation and purification.
Method 1: Using Enzymes
4. The centrifuge pellets was resuspended in 500uL of TE buffer till no visible clumps is seen.
5. 1mL of lysozyme is added to the resuspended cells. The purpose of this is to lyse the cell wall so that the protein would now be in the supernatant. It was left to stand for 15 minutes.
Method 2: Freezing and Thawing
6. The tube is placed in liquid nitrogen (-196◦C) till the solution freezes and was immediately thawed in hot water. The process was repeated a total of 3 times. This would cause mechanical stress to the cells as they expands when freezed and contracts when thawed.
Video: Freezing and thawing of cells
Method 3: Sonication
7. The cells implode under the process of sonication where they are exposed to ultra sonic waves which produces vibrational pressure. Since there would be heat produced, the process was conducted in a ice bath to prevent denaturing of proteins.
8. The process was repeated for 4 cycles, each cycle of 25 seconds and rest of 10 seconds during intervals.
Video: Shows the process of sonication
9. After sonication, the cells contents are now in the supernatant. The tube is then sent for centrifugation at 10,000rpm for 20 minutes. The GFP should now be in the supernatant.
Stage 2
10. The supernatant would then be purified by gel permeation chromatography. The column used is a polymer gel resins (Sephadex G75). The resins has very small pores, therefore the small molecules would spend more time in the resins compared to the bigger molecules. The GFP would then be separated according to size.
11. The buffer used is ammonium bicarbonate, where it is used to flush out the proteins. It is then collected in fractions to be analysed by taking OD readings.
Video: How gel permeation is conducted
The samples collected and 1.5mL of fraction was transferred to a cuvette to be read by the spectrophotometer at 476nm.
The chromatogram is shown below.
The absorbance readings were carried out at 476nm, which is the wavelength at which GFP maximally absorbs at and gives out its usual fluorescence, this protein will be easily detected by the spectrophotometer. The chromatogram illustrates the efficiency of the separation of GFP protein from the sample. Based on the graph, there are 3 peaks. Besides GFP, 2 other proteins were detected. These 3 proteins are not well separated as the peaks overlap. Hence, the “peak” fractions will be pulled together and further purified by other methods like affinity chromatography, where the separation is very specific. Since the number of proteins in the cell free extract is numerous, GFP is unable to be identified. It is likely that the fractions contain a large portion of contaminating proteins as these proteins have a similar size to GFP. Hence, all the 3 “peak” fractions should be collected and further purified. This includes pooling fraction 2 and 3, and collecting fraction 5 and 7.
