failed a test paper?

Day 2: Agar plate results

Tuesday, January 6, 2009

Hey guys,we are back again.This is our day 2!!

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)

From the results obtained, we can see that the growth of E.coli is not well developed as there is only a small portion of growth found. The possible explanation for poor growth could be that the E.coli were already dying and thus the colonies are weak. Therefore, there were lesser proliferation of E.coli colonies.

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.

♥8:03 PM

-Questions and Answers

Experiment 1

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

The differences are summarized in the following table

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.

The fermentation process includes the upstream processing, the actual bioreactor and the downstream processing. The upstream processing includes all the activities required to convert the crude raw materials into processed raw materials which can be fed into the bioreactor. This includes 3 main activities – Media, Cell line, and Equipment preparation. The media is must be optimized and formulated so as to allow the cells to produce out the desired product. Media is required to support cell growth and also to sustain the cell metabolism. The media needs to be sterilized and purified to ensure that the conditions in the bioreactor are kept sterile, where no contaminants may grow. Equipment is also sterilized and cleaned to ensure that the previous bioprocess leaves no residues behind which may contaminate the subsequent bioprocess. Cell line needs to be prepared, where the cells are allowed to grow to optimum growth condition. As some of the cells need to be induced to produce products, this seed culture step can help to induce the production of the desired product. This helps to reduce the lag phase and also obtain a pure culture for the scale-up fermentation process.
During the bioprocess, process conditions needs to be monitored and optimized for the cells to grow and produce products.
The downstream processing includes the harvesting, isolation and purification of the product. These steps will generate the final product to be delivered to the final consumers.
This is illustrated by the following diagram.

Experiment 2

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

Bactotryptone is as a Carbon, Nitrogen and Sulphur source.
Yeast extract is a Carbon, Nitrogen, Sulphur source and contains vitamin B complex.
NaCl provides with Na+ for membrane transport and helps in the maintenance of osmotic equilibrium.

1b. What is the purpose of ampicillin?

The purpose of ampicillin is to prevent contamination.

1c. Why is ampicillin added only after autoclaving?

Ampicillin is only added after autoclaving because it is heat sensitive and will be inactivated due to heat.

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

The calibration of the pH probe is the relation of the pH probe and the units of pH.

2b. Why is hydrochloric acid not suitable as a correction agent for pH?

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

2c. What is meant by polarization of the pO2 probe?

The polarization of the pO2 probe is to place the pO2 probe into the culture media and to aerate with nitrogen.

2d. What is a peristaltic pump?

Peristaltic pump is a pump to ensure the direction flow of fluids is correct.

3a. What is the purpose of arabinose?

The purpose of arabinose is to induce the production of Green fluorescent protein (GFP).

3b. Describe the sterile techniques used in seed preparation.

The sterile techniques include working in the area near the flame. Unnecessary movements are avoided to ensure that the contaminants will not move into the working area.

3c. Why do we perform step-wise scale-up instead of transferring directly to the fermenter?

By carrying out a step-wise scale-up, the cells will be allowed to grow to the optimum growth phase before the actual inoculation to the fermenter. This helps in the reduction of the lag phase. The cells can also be induced to produce the desired product. Through a step-wise scale-up, conditions of the process can also be optimized.

Experiment 3

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

The control philosophy for pH, temperature and dissolved oxygen levels are controlled by a feedback system. This is where the measuring instrument will detect a change in the environment and send a signal out to the control system. The control system will then determine the set point to which the parameter needs to be altered to return to the pre-determined value. The signal will then be sent to the control element to make the necessary alterations such as the opening of a valve.

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

Spectrophotometer makes use of Beer-lambert’s law. The Beer-lambert’s law states that the absorbance is directly proportional to the concentration of the absorbing material in the solution. This illustrated by the following equation:
A = Elc
where A is the absorbance measure
E is the extinction coefficient
l is the path length or thickness of the cell containing the sample
and c is the concentration of the absorbing material.
600nm is used because this is the wavelength at which the cellular components are maximally absorbed at.

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. GFP should be harvested after the stationary phase and before the death phase. The product was harvested during 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.

Some advantages of using a computer control system include the automatic data logging, where data is collected and stored. Data analysis is also automated, where the mathematical models of the process is calculated. The computer can also help in the optimization of the bioprocess, where the process is controlled by the signaling to devices such as pumps, valves, switches and alarms.

The history chart is shown in the diagram above.
From the history chart, it is observed that the changes in conditions were quickly resolved as the peaks were sharp, having a steep gradient. The gradient indicates the speed in which a deviation from the set point is returned to the pre-determined value.

Experiment 4

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

The results are shown below:

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.

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?

A protein of 50,000 kDa will elute out first as it is larger than GFP of a 27,000 kDa. The larger the protein, the lesser time will it spend in the column as the large protein is unable to enter the small pores of the stationary phase. Hence, it will elute out with the mobile phase first before GFP is eluted out.

References

http://en.wikipedia.org/wiki/Calibration
http://www.sigmaaldrich.com/etc/medialib/docs/Fluka/Usage/lb_agar.Par.0001.File.tmp/lb_agar.pdf