Navigate through the website by clicking on the icons at the top.
Now that we have mastered incredible GFP technology, our next stop: global domination & world peace!
2. The pH probe, pO2 probe, foam and level probe were installed into the top plate. Necessary adjustment of the height for the foam and level probe is carried out when necessary. 4. The exhaust condenser, air inlet and exhaust filters and manual sampler unit were installed. Further questions... (a) What is meant by calibration of the pH probe? Calibration of the pH probe is meant to set the pH point to be uniformed within the fermenter. Hence, a slight change in pH can be detected by the pH probe. (b) Why is hydrochloric acid (HCl) not suitable as a correction agent for pH? (2) For the sterilization of the bioreactor: 2. All silicone tubings except for exhaust filter and female STT coupling of sampling unit were clamped. All filters and sockets were covered with aluminium foil to protect from condensing moisture. 3. The bioreactor was autoclaved with steam at 121 degree celsius for 20 minutes. 4. The pO2 electrode was polarized for at least 6 hours and calibrated by aerating with nitrogen. 5. The addition lines were connected to peristaltic pumps. 1. Luria-Bertani (LB) medium was used for both seed-culture and fermentation media. 2. 1000ml of distilled water was added to 25g LB-powder containing bacto-tryptone, yeast extract and sodium chloride (NaCl). 3. The pH was pre-equilibrated in the powder; therefore, no equilibration was needed. 4. The media was autoclaved at 121 degree Celsius for 20 minutes. 5. Ampicillin was added to the final concentration of the media once the broth had cooled to below 50 degree Celsius. 6. The media was stored at 4 degree Celsius till inoculation. Further questions... (a) Bacto-tryptone (b) Yeast extract It provides any type of organic molecule that a growing bacteria would need and essential for cellular metabolism. Glutamate contributes as an intermediate in processes such as glycolysis, gluconeogenesis and citric acid cycle. Vitamins are bio-molecules that act as catalysts and substrates in chemical reaction. (c) NaCl (d) dH2O (e) pH (ii) What is the purpose of ampicillin? (iii) Why is ampicillin added only after autoclaving? (4) For the preparation of seed culture: 1. The pGLO transformed E. coli was streaked onto a LB/Amp (ampicillin)/Ara (Arabino) plate and was incubated for 24 hours. 2.After incubation, several colonies were obtained and transferred to the flask containing 100ml LB medium with ampicillin. 3. The flask was left in shaking incubator and incubated at 32 degree Celsius for 24 hours or at r.t.p for 48 hours. This is to facilitate the purpose of scaling-up. Further questions... (a) What is the purpose of arabinose? (b) Describe the sterile techniques used in seed preparation. (c) Why do we perform step-wise scale-up instead of transforming directly to the fermenter?
Day 1Experiment 2: Equipment, Media and Seed Culture Preparation
Objectives
1. To describe the steps to prepare a bioreactor2. To prepare the media for seed culture and scale-up fermentator3. To prepare seed culture for scale-up fermentation (1) For the preparation of the bioreactor:
1. The pH electrode was calibrated using standard buffer solution. .jpg)
3. The addition agent lines for acid, base and antifoam were connected and the levels in the storage bottles were checked. 5. The water jacket that was filled with water was checked. HCl will cause corrosion of the metal parts of the fermenter when it accidentally leaks out from the tubing which results in spoilage of the fermenter. (c) What is meant by polarization of the pO2 probe?Polarization of the pO2 probe meant that the oxygen level measured is only the measurement of the tested solution and not the measurement with that present in the electrolyte solution. (d) What is peristaltic pump? Peristaltic pump is apparatus consists of flexible tube inside a circular pump casing and a rotor with a number of rollers that are attached to the external ciircumference compresses the flexible tube. The rotor ensures that the fluid to be pumped is moving through the tube.1. All cables except the temperature probe were disconnected..jpg)
.jpg)
(3) For the preparation of media:.jpg)
(i) Explain the purpose of each ingredient found in the LB media.
Ingredients that are found in the LB media are:It is an enzymatic digestion of the protein to provide amino acids to the growing bacteria.NaCl provide sodium ions which are used for transportation and maintainig osmotic balance.The purpose of adding water is to suspend the solids.pH at 7.5 is optimal for cell growth.The purpose of ampicillin is to use it as a selective tool to allow growth of resistant E.coli and also to prevent other growth of other bacteria (lower the risk of contamination within the media). It acts as a inhibitor of enzyme transpeptidase in the synthesis of cell wall by gram positive bacteria and hence only allowing ampicillin resistance bacteria to grow.The temperature of 121 degree Celsius during autoclaving will cause the ampicillin structure to be denatured. This will cause ampicillin to loss its function.The purpose of arabinose acts as the regulatory control for the expression of the GFP gene. Hence, when bacteria were plated onto the LB agar containing arabinose, this will apear bright green. If arabinose is absent, colonies will appear white as the gene is turned off. The sterile technique used in seed preparation is the adition of ampicillin to kill of unwanted bacteria that may be present in the agar.This is to prevent longer lag phase for the bacteria to multiply. This is because bacteria will take time to adapt to its new environment. The action of adding bacteria directly to the fermenter may kill them as it will give "shock" to the bacteria.