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.
- To carry out scale-up fermentation process to increase the yield of desired protein product (Green florescent protein in this case).
- To monitor cell growth and product formation through manual sampling and computer data logging.
The bioreactor was prepared in the following steps:
1. LB medium was added into the bioreactor.
2. pH probe was calibrated and installed together with pO2 probe and foam probe.
3. Bioreactor was connected to addition agent lines for acid, base and antifoam.
4. Exhaust air filters, condenser and other accessories were connected and the water jacket was filled with water.
5. All tubes were disconnected, with expection of temperature probe and was sent to autoclave at 121 degrees celsius for 20 minutes. All filters and sockets were covered with aluminium foil to protect from condensation on them.
6. pO2 probe was polarised and calibrated.
7. All the tubes were connected back and the pumps for reagents were switched to auto mode.
8. Penicillin and arabinose was added into the reactor after autoclaving as they are heat sensitive.
9. The seeded culture was then transferred into the reactor and the scale-up fermentation begins.
10. The following parameters were set by the control panel
- pH 7.5
- stirred speed of minimum 10%, maximum 90% and set to auto control.
- pO2 set point of 20% (auto), stir to CASC and airflow to CASC
- Airflow of minimum 25% and maximum 100%
However, a sample of medium was taken before inoculation of cells into fermentor which serves a blank for spectrophotometer reading.
The videos below show how ampicillin was added into the bioreactor and how the seeded culture was added, and lastly how the pump works to add the reagents.
Video 1: How ampicillin is added
Video 2: How is seeding of cells done
Video 3: How the pump adds base to bioreactor
After the seeding of cells is done, the first sample was retrived from the bioreactor. The subsequent samples were taken hourly from 9am to 6pm.
Objectives
- To describe the steps to prepare a bioreactor
- To prepare the media for seed culture and scale-up fermentation
- To prepare seed culture for scale-up fermentation
Hey friends so, after the streaking of colonies onto the agar plate on the 1st day,we obtained the results of the streaks.
(clockwise direction: the 1st 2 at the top -TSO, the bottom plate on the right - Our group)
After viewing the results, we did a scale-up of the cells from the agar plate. A small amount of E.coli cells were obtained from the plate and added into the shake flask. After which it was incubated at 32 degree celcius for 24 hours.
The video below shows the procedure of how the seeding of cells was done aseptically.
Step 1: Take a loopful of E.coli from agar plate.
Step 2: Put into the seeding flask and incubate at 32 degrees Celsius for 24 hours.
We will learn how to extract GFP (Green Fluorescent Protein) from E.coli. Does this sound INTERESTING?? Well of course it does, so come read our blog and have a good time.
Enjoy!!
Objective of Overall experiment
To extract GFP from E. coli cells.
Objectives for part 1
- To be familiarize with the parts and components of microbial and mammalian bioreactors
- To be introduced to the basic operation procedure of a bioreactor
Today, we had our very first bpt prac of the semester with Ms Ang. It was kind of intersting as we were taught how to do and apply scale up. Below are some of the steps that we did for the initial stages of the scale up.We learnt many interesting things about how scale up and the initial stages of scale up was done.
For the first part of the prac, we learnt how what the bioreactor was and how it looked like, as well as the functions and purposes of each component of the fermentor. Here are some pictures of the fermenter and also a pictorial form of the fermenter where a short explanation of the function is given.
The steps for extracting samples out from fermenter aseptically.
Step 1: Make sure all the clamps on the tubes 1 and 3 are clamped tightly before proceeding.
Step 2: Unclamp tube 1 and pull the syringe to extract out the media from the fermenter.
Step 3: After a certain of volume is extracted in the sampling tube, push back the syringe to prevent having any dead volume of media trap in the tube. As it may give inaccurate result when another sample is withdrawn.
Step 4: Clamp tube 1 after no more volume is present in the tube.
Step 5: Remove the syringe from the tube and add volume to the syringe. Connect the syringe and tube before unclamp tube 3 is done.
Step 6: Depress the syringe completely to extract out the volume present in the sampling tube into a test tube and clamp tube 3.
The 2nd part of the prac was used to make Luria-Bertani(LB) broth medium. Below are some pics as to how it was made by our group.
LB broth is used to seed the culture as well as fermentation media. The following steps are performed to prepare the 2L medium.
