Geeky Corner - Answers to Further Questions

Experiment 1 – Familiarization with the Bioreactor and its operation

Aninteractive four-gas controller is recommended to enable proper balance of each gas component to assure proper pH and D.O. control.

Dissolved Oxygen - Most microbial and mammalian cells require oxygen to support their metabolism. Air, the most commonly used source for oxygen, may be added to the culture medium either by direct sparging, indirect gassing or gas overlay. Microbial fermenters are rated for addition of up to 2 volumes of gas per volume of liquid per minute (VVM), while mammalian bioreactors are usually rated up to 0.5 VVM. Consequently, the airflow control capabilities of the mammalian cell culture bioreactor must be more refined than those for microbial culture.

Shear Sensitivity - Microbes have very robust cell walls (Figure 1), allowing direct gas sparging without fear of reduced cell viability due to shear forces exerted by contact with gas bubbles. Agitation rates in microbial fermenters are often set to 800 rpm or more, providing both ample oxygenation and homogenous mixing of viscous cultures. The preferred microbial impellers are radial type, such as the Rushton turbine impeller.

Foam - Direct sparging with gas and a high protein content of the medium are the two main culprits in foam production. The addition of antifoam agents via a peristaltic pump controlled by a foam sensor in the culture medium is common for microbial cultures, but is usually not considered in mammalian cell culture. The Cell Lift impeller also features a foam-elimination chamber and an optional Air Wash SystemTM to minimize foaming.

Study the work flow on page 1 of your laboratory manual. Describe the typical activities that are performed for each stage in the fermentation process.

1)Familiarization with the Bioreactor and its Operation (Exp 1)
2)Equipment, Media and Seed Culture Preparation (Exp 2)
3)Inoculation, Fermentation and Monitoring (Exp 3)
4)Isolation and Purification of Product (Exp 4)

1st stage:
-Different parts of the fermenter and their function were introduced.

2nd stage:
- Preparation of the (Luria-bertani medium) was done.
- Preparation of the bioreactor was done by calibrating the pH electrode, installing the pH probe, pO2 probe, foam and level probe, installing of exhaust condensers, air inlet and exhaust filters.
- Preparation of seed culture is done by using the streaking method on the plate of E.coli on LB/Amp/Ara plate.

3rd stage:
- Ampicilin and arabinose was added to the agar plate containing the culture medium. (Final concentration of adding is 0.2%)
- Control parameters of the fermenter are set to a temperature of 32 degree Celsius.
-Seed culture cells are inoculated into the fermenter and 10ml of the sample are taken out every hour. (11 test tube were prepared for the sample to be taken out)

4th stage:
- Isolation of the green fluorescent protein was done.
- Purification of the extract was done using the gel filtration.
- Absorbance readings of the extract were taken using the spectrophotometer.


Exp 2 – (Part A) Procedure for preparation of equipment, media, and culture.

Experiment 2A – Further Questions:

1) On media preparation:

a. Explain the purpose of each ingredient found in the LB media.

The ingredients found in the LB media are Bacto-tryptone, yeast extract, NaCl, dH2O and pH.

-Bacto-tryptone is used to provide essential

amino acids to the growing bacteria

-Yeast extract is used to provide a plethora of organic compounds helpful for bacterial growth, Vitamins and certain trace elements

-Sodium chloride provides sodium ions for transport and osmotic balance

-Distilled water to suspend the solids to dissolve it

-Prior to autoclaving, some labs adjust the pH of LB to 7.5 or 8 with sodium hydroxide. The downside of using sodium hydroxide is that the pH will not be buffered which means that the bacteria will rapidly change the pH as they grow. To get around this some labs prefer to adjust the pH with 5-10 mmol/l TRIS buffer diluted from 1 mol/l TRIS stock at the desired pH.

b. What is the purpose of ampicillin?

Ampicillin is an antibiotic; antibiotics prevent bacteria from growing. As pGLO transformed E.coli is resistant to ampicillin, the purpose of ampicillin is to prevent other cells/ unnecessary bacteria, which do not possess the resistance, from growing/ reproducing. Only the desired organism is allowed to replicate.

c. Why is ampicillin added only after autoclaving?

Ampicillin is very sensitive to heat. If ampicillin was to be added before autoclaving, it will denature the structure of the ampicillin especially after autoclaving 100ml of LB medium at a high temperature of 121oC for 20 minutes. This is because the bonding strength of ampicillin is weak and it is easily broken up by high heat. Therefore, it is necessary for ampicillin to be added only after autoclaving.

2) On equipment preparation:

a. What is meant by calibration of the pH probe?

Calibration of the pH probe is to use buffer solutions (pH 7 and pH 4 or 9 depending of on the culture) to make sure that the fermenter is of optimum pH for scaling up of culture.

b. Why is hydrochloric acid not suitable as a correction agent for pH?

Hydrochloric acid is not suitable as a correction agent for pH because it is a strong acid and highly corrosive liquid. HCl can dissociate or ionizes practically completely in water. It is strong enough to removes water from the solution in the fermenter and thus affect the optimum composition of the fermenter.

Exp 2 – Part B Procedure for preparation of media, and culture.

Experiment 2B – Further Questions:

3. On seed preparation:

a. What is the purpose of arabinose?

The purpose of the arabinose is to ‘switch’ on the cells that have inherited the pGLO gene that codes for the fluorescent protein, and cells which inherit the pGLO genes when grown in the arabinose medium would produce a fluorescent colour.

b. Describe the sterile techniques used in seed preparation.

The pGLO transformed E.coli was obtained from -800C freezer. Under such a low temperature, many microbes could not able to survive. Thus the contamination from the storage of the cells is reduced significantly. Another technique that we used is that we streak the bacteria on the ampicillin medium. It inhibits the growth of the bacteria and allows the growth of the E.coli that has successfully taken up the pGLO, which would encodes for the ampicillin resistant gene. And when we transfer the colonies of the pGLO transformed E.coli into the 100ml LB medium with ampicllin, we used sterile techniques which is wearing gloves during handling the material which would prevent any contamination from the hands and work in the fume hood.

c. Why do we perform step-wise scale-up instead of transferring directly to the fermenter

This is to ensure that it is the specific bacteria we want, which will produce the GFP, and that there is no contamination with other microbes. However, if the bacteria culture is transferred directly into the fermenter, there may be contaminants or other microbes being transferred in too, due to the bacteria culture not being pure.

Experiment 3 - Inoculation, Fermentation and Monitoring I

Further Questions

1) The pH level is controlled by the release of the acid and base as cells in the fermentor are sensitive to drastic changes in pH. If the pH of the solution in the fermentors is too low, then an appropriate amount of sodium hydroxide (base) will be released to neutralise the pH. Likewise if the pH of the solution in the fermentors is too high, then an appropriate amount of Sulphuric acid will be released to neutralise the pH.

The temperature in the medium is measured using a temperature probe located in the fermentor. Should there be any noticeable increase in temperature; cool water will enter via the cooling water inlet of the cooling jacket. The water will decrease the temperature of the solution in the fermentor by absorbing the heat. The water then leaves via the cooling water outlet. If the temperature in the medium is too cool, then warmer water will enter into the cooling jacket instead.

Dissolved oxygen is required by the growing cells for respiration. The amount of dissolved oxygen in the medium is measured using a dissolved oxygen probe located in the fermentor. If the dissolved oxygen level is too low, air (oxygen) will be pumped to the sparger so as to introduce more dissolved oxygen into the media.


2) The spectrophotometer is a device that is used to measure the concentration of the sample. This is done by the following mechanisms: Light is emitted form the spectrophotometer and split into a spectrum. The appropriate slit lets through the specific wavelength of the light. The filtered light is absorbed by the substance in the sample (GFP) and the detector will detect the light absorbed by the sample.

Thus if the detector detects a low absorbance (abs), then it can be confirmed that the substance is of lower concentration as lesser light is absorbed. This indicates that the cell density (OD) is lower. If the detector detects a high absorbance, then the substance is of a higher concentration as more light is absorbed. This will indicate that the cell density is higher.

The reason why 600nm was used was due to the fact that the GFP had a peak emission of >500nm, around 509nm. If a <600nm>was used was due to the fact that the GFP had a peak emission of >500nm, around 509nm. If a <600nm>

Experiment 4: Isolation and purification of product
Answers to further questions

1) By looking at the graph, it shows that the highest absorbance reading was at fraction 2. The highest absorbance shown on the graph means that most of the green fluorescent protein we collected was in this tube. There were not many changes before fraction 2 because the samples have just been added and therefore it takes time for it to reach the end of the column. After fraction 2, the absorbance readings started to decreased at a very fast rate. This is because at the highest peak, GFP had been removed by the solvent, though, the second graph show a slight increase in the fifth fraction which may be due to contamination during harvesting of sample or collecting of the purify solution.

2) The protein with a Mr of 50,000 kD would most likely elute in a fraction before GFP. Higher Mr give rise to heavier molecular weight,heavier therefore sinks faster than GFP with lower Mr and lighter molecular weight.