Experiment 1



1. State the differences you observe between a microbial bioreactor and a mammalian cell bioreactor.

Differences between a microbial bioreactor and a mammalian bioreactor:

Microbial bioreactor
Impellers spinning at higher speed

Mammalian cell bioreactor
Impellers spinning at lower speed.

Mammalian cells are more susceptible to agitation than bacterial cells. Thus, a slower impeller speed is needed to ensure that the cells are not killed.

2. 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.

Workflow and activites

Familiarization with Bioreactor and its operation
Labeling of the bioreactor and the function of each of the components.



Equipment media and seed culture preparation
Preparation of media, bioreactor and seed culture.



Inoculation, fermentation and monitoring
- Inoculation of medium with seed culture.
- Taking hourly samples.
- Recording the absorbance readings.



Isolation and purification of product
- Isolation of GFP(disruption of cell using enzymes, freezing-thawing and sonication method.
- Purification (by means of gel permeation chromatography)

Experiment 2

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

Bacto-tryptone is a source of amino acid needed for bacteria growth. Yeast extract provides the vitamins needed and sodium chloride keep the broth at a certain ionic strength.

b) What is the purpose of ampicillin?

Its purpose is to prevent contamination by undesired microorganisms.


c) Why is ampicillin added only after autoclaving?

The heat will inactivate the antibiotic, if added before autoclaving.

2 a) What is meant by calibration of the pH probe?

It is to establish the unit of the pH probe that it is going to measure so that it can measure that pH of the solution accurately.

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

Hydrochloric acid is too corrosive as a correction agent for pH.

c) What is meant by polarization of the pO2 probe?

It means to equilibrate the partial pressures of O2 between the environment and the inside of the sensor membrane.

d) What is a peristaltic pump?

A peristaltic pump is to pump fluids into the fermenter. Examples would be adding acids and bases when needed.

3 a) What is the purpose of arabinose?

The pupose of arabinose is to induce the production GFP and is a carbon source for the bacteria.

b) Describe the sterile techniques used in seed preparation.

The inoculating loop to streak the bacteria was sterilized over the Bunsen flame before inoculation. The spreader used to spread the bacteria on the agar was sterilized over the Bunsen burner before spreading. When transferring to the flask containing 100mL LB medium with ampicillin, it was worked near the Bunsen flame, within the sterile field to minimise contamination.

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

E.coli needs to adapt to the culture conditions of the fermenter. The organism may not do so if transferred directly as the environment of the fermenter is different from the agar. Also, any amount of media would need a minimum level of inoculum to allow proliferation.

Experiment 3

1. Explain the control philosophy for pH, temperature and dissolved oxygen as was used in the fermentation process.

pH- When the E.Coli is growing in the medium, the pH of the media will drop as cellular respiration takes place. The pH probe will detect changes in the pH of the solution and send a signal to the computer. In order to maintain the optimal condition for the growth of E.Coli, the peristaltic pump will pump base into the medium to increase the pH back to the optimum level.


Temperature- Stirring and respiration of the E.Coli will produce heat in the medium. The temperature probe will measure changes in the temperature and send data to the control system. The control system will signal the cooling jacket to cool the fermenter. The cooling jacket cool the fermenter by pumping water in from one end and out from the other end, taking away the excess heat in the process. Both rate of water flow and temperature of water can help in the cooling process.


Dissolved oxygen- When the E.coli grows, oxygen levels are reduced in the media. The dissolved O2 probe will measure the dissolved oxygen levles and send the data to the control system. The control system will then send signal to increase sparging, thus increasing the O2 levels.

2. Describe the principle of the spectrophotometer which was used to determine the cell density (OD600). Why was 600 nm used?

The spectrophotometer measure the amount of light that is left after the starting beam is transmitted through a sample. However, it converts this to the amount absorbed as absorbances values to facilitate the technician who just needs the amount of light absorbed. 600nm wavelenght was used in measuring cell density because it is the wavelength that the cell absorbs light maximally.

3. Is GFP a primary or secondary metabolite? At which phase should the product be harvested? At which phase was the product actually harvested?

GFP is a secondary metabolite as the cell can still survive in the absent of GFP. The phase should be harvested at the end of the secondary phase. However, the product was harvested at the death phase.

4. What are some advantages of using a computer control system? From the history chart (which will be given to you by your supervisor after the fermentation), comment on the effectiveness of the computer control.

Computer control system make the adding of pH and the adjustment of temperature automated so that the environment of the fermemter can be controlled more effectively. In addition, it helps to collect and store data so that the technician can interpret the results obtained. Other advantage includes data analysis by performing calculation based on mathematical models.

The history plot clearly shows the effectiveness of the computor control. Both the temperature and pH stayed at nearly a constant level. The most convincing evidience, howerver, would be the changes in the dissolved oxygen content and the stir rate. The plot clearly shows the rises and drops of oxygen levels during the phases of cell growth. The stir rate is shown to be compensating for this fact by increasing speed when there is low oxygen, and decreasing speed when there is high oxygen content.

Harvesting

1. Plot a graph of your A476 absorbance values (Y-axis) vs fraction number. Comment on your chromatogram.

Absorbance readings of the purified Green Fluorescent Proteins (GFPs):

From the graph above, we observe that the absorbance reading for the 8 fractions were close to 0.00 with the exception of fraction 2, at between 0.1 and 0.2, which is then observed to have a steep drop at fraction 3. This is most likely due to the fraction having peak absorbance containing materials, in this case GFP. This shows that the GFP is large enough to be eluted rather early. Since the other fractions have almost zero absorbance, most of the GFP would be in the earlier fractions of 2 and 3. This means that the GFP is pure as only they are left in those fractions while the other proteins are in the other fractions.

2. GFP has a Mr (molecular weight) around 27,000 kD. Though we were unable to see them, the cell free extract also contained hundreds or even thousands of other proteins. Do you think a protein with a Mr of 50,000 kD would elute in a fraction before or after GFP? Why or why not?

It will elute before GFP. This is because large molecules spend less time in the gel permeation chromatography column.

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