Further Questions- Expt 1 and 2

Experiment 1

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

click on the table to have a clearer view.

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.

Getting to know the parts of the bioreactor (exp 1)

To familiarize with the bioreactor and know its’various parts and components. Not only that, to know the functions that each part carries. To also learn how the bioreactor works and how it is fixed and which are the components it is connected to.

Equipment, Media and seed Culture Preparation ( Expt 2 )

Setting up of the equipment needed, the bioreactor by sterilising and making the media needed for the fermentor. Seed culture where the desired product needs to be cultured and grown to required state. The desired product is the pGLO transformed E.coli which is E.coli with a glowing gene that causes the bacteria to glow when seen under UV light. Media preparation is done with the required amounts of ingredients and then used to be placed in the bioreactor. The media is very important because the ingredients used is vital in sustain the growth of the cells as it acts like a nutrition broth.

Inoculation, Fermentation and Monitoring (Expt 3)

The Inoculated pGLO transformed E.coli was placed in 100ml of broth and left to inoculate and then placed in the fermentor for further inoculation for fermentation. This fermentation is done to provide the various requirements for the cell to grow and for scale-up. After the fermentation is done the samples are collected hourly for a day and the media is monitored. Monitoring is done by taking the absorbance reading of the samples and calculating the fermentor parameters.

Isolation and Purification of Product (expt 4 )

The first stage done for Isolation is to lyse the bacteris cells so as to release the proteins. Since all the cells are in the media, to obtain the cells centrifugation is done and the cells would be pelleted at the base of the tube. After discarding the unwanted supernatant and resuspending the pellet in the buffer lysozyme was added so as to activate the enzymatic digestion of the cell wall. This is followed by freezing and thawing the cells in liquid nitrogen so as to add mechanical stress to the cell wall. Lastly, the final step of cell disruption is to perform sonication which causes the cell wall to break under vibrational pressure. After all this is done, the contents are centrifuged one last time and now our desired prouct is in the supernatant.

Finally the purification is done by size exclusion chromatography to obtain our desired pGLO transformed E.coli. These results are then viewed under the UV light and confirmed whether the E.coli has been transformed.

Experiment 2

1. On media preparation:

a. Explain the purpose of each ingredient found in LB media.
The ingredients found in LB media are Bacto-tryptone, Yeast extract, NaCl and dH2o.

Ingredient	Purpose
Bacto-tryptone	For providing essential amino acids to the growing bacteria
Yeast extract	Provides vitamins and certain trace elements
NaCl	Provides vitamins and certain trace elements.
dH20	Provides the solvent for the media.

b. What is the purpose of ampicillin

Ampicillin is an antibiotic and an antibiotic inhibits the bacteria growth. pGLO transformed E.coli will encode for ampicillin resistance. Thus, only the transformed E.coli will be able to survive on the medium which contains ampicillin. If the E.coli does not contains the plasmid, pGLO, it will not be able to survive on the medium.

c. Why is ampicillin only added after autoclaving?

The autoclaving of the LB media will destroy the microorganisms present in the media. Ampicillin is added only after autoclaving is to ensure that other microorganisms will not contaminate the media after autoclaving.It is meant by correlating the voltage produced by the probe ( approximately0.66 volts per pH unit ) with the pH scale.



2. On equipment preparation:


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

Calibration of the pH probe refers to checking and adjusting the pH to the appropriate value. When the pH is low, it is too acidic or too much H+ ions are present, base will be added to adjust the pH. On the contrary, when the pH is too high, the amount of H+ ions is low, thus acid will be added.



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

Hydrochloric acid(HCl) is not suitable as a correction agent for pH because it is a very strong acid and by adding a small amount of acid, it can create a big change in the solution.


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

Polarization of the pO2 probe refers to the voltage applied across the electrodes of the dissolved oxygen (DO) probe. At the anode, the silver ions are oxidized producing silver chloride and electrons whereas at the cathode, oxygen is reduced and accepting the electrons and produce hydrogen ions. The current is generated by the chemical reactions conducted through the filing solution. This current is proportional to the oxygen concentration and hence, converted by the meter to a DO reading.

Polarization is a term to describe when the probe has reached equilibrium and able to produce stable and reliable measurements. To polarize the probe, voltage must be continuously applied to the probe thus allow the oxygen reaction to proceed. When the voltage stops, the reaction cannot continue and thus the probe is no longer consider polarized.


d. What is a peristaltic pump?

A peristaltic pump is a type of postivie displacement which is used for pumping a variety of fluids. There is a flexible tube inside a circular pump casing and there will be fluid contained in it. There is also a rotor attached to the external cirumference and compresses the flexible tube. Hence, when the rotor turns, part of the tube will be under compression and closes, forcing the fluid to get pumped out of the tube. When it is back to its natural state or not under compression, the fluid flows back to the pump.

This is used in fermentation because it can pump clean or aggressive fluid and contamination cannot occur easily.



3. On seed preparation:

a. What is the purpose of arabinose?

Arabinose is used as an inducer in the production of Green Fluoroscent Protein (GFP). It induces the cell to produce more GFP when the cell comes in contact with the arabinose. thus, express the protein. Hence, the bacteria will be able to glow.



b. Describe the sterile techniques used in seed preparation.

During seed preparation, the inoculation of the bacteria should be done near the flame of Bunsen burner. The inoculating loop must be flamed to sterilize it and the spreader used to spread the bacteria to be soaked in ethanol first, allowed to dry and flamed then spread the bacteria on the agar plate.


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

Step-wise scale-up is performed instead of transferring directly to the fermenter as step-wise scale-up allows the bacteria to adapt the culture conditions. Another reason is that it is possible to monitor the optimum growing conditions as compared to large scale medium.