failed a test paper?

Day 1: Understanding of bioreactor

Monday, January 5, 2009

Hey Friends!! Welcome to our site.

We will learn how to extract GFP (Green Fluorescent Protein) from E.coli. Does this sound INTERESTING?? Well of course it does, so come read our blog and have a good time.
Enjoy!!

Objective of Overall experiment
To extract GFP from E. coli cells.

Objectives for part 1
- To be familiarize with the parts and components of microbial and mammalian bioreactors
- To be introduced to the basic operation procedure of a bioreactor

Today, we had our very first bpt prac of the semester with Ms Ang. It was kind of intersting as we were taught how to do and apply scale up. Below are some of the steps that we did for the initial stages of the scale up.We learnt many interesting things about how scale up and the initial stages of scale up was done.

For the first part of the prac, we learnt how what the bioreactor was and how it looked like, as well as the functions and purposes of each component of the fermentor. Here are some pictures of the fermenter and also a pictorial form of the fermenter where a short explanation of the function is given.

Motor: Use to spin the impeller

Impeller: There are 3 impellers and 6 blades. For agitation and aeration of media.

Sparger: To introduce air into the fermenter

Baffles: There are 4 baffles. Use for better mixing and prevent whirpool effect.

Inlet air filter: To provide sterile air

Condenser: To condense water vapour and prevent clogging of air filter.

Outlet air filter: Sterile air, if media is toxic

Cooling jacket: Maintain temperature of media

Rotameter: To measure flow rate of air into the reactor.

Pressure Gauge: To measure pressure in the fermenter.

Temperature Probe: To measure temperature of medium, but it does not regulate the temperature.

pH Probe: To measure pH of the medium

Dissolved Oxygen Probe: To measure oxygen content

Level Probe: To measure level of medium, which important in fed-batch especially if the fermenter is made of stainless-steel.

Foam Probe: To measure foam level and antifoam is added into the fermenter via computer. This is to prevent foam from clogging the air filter.

Acid: Sulphuric acid is used in this experiment , as hydrochloric acid is to corrosive. It is added when the pH level is too alkaline.

Base: Sodium hydroxide is used in this experiment and is added when the pH level is too acidic.

Antifoam: Antifoam is added to break foam.

Sampling Tube: The tube is used to take sample aseptically.

Control Panel: It is used to keep the conditions of the fermenter at optimum level.

We even had a hands-on experience with the fermentor. The video shows how we extract samples from the fermenter aseptically.

In the video there are also explanation why some steps are taken such as, pushing the culture back to the ferementer so that there won't have a stagnant volume at the tube connecting the fermenter to the sampling tube.

The steps for extracting samples out from fermenter aseptically.

Step 1: Make sure all the clamps on the tubes 1 and 3 are clamped tightly before proceeding.

Step 2: Unclamp tube 1 and pull the syringe to extract out the media from the fermenter.

Step 3: After a certain of volume is extracted in the sampling tube, push back the syringe to prevent having any dead volume of media trap in the tube. As it may give inaccurate result when another sample is withdrawn.

Step 4: Clamp tube 1 after no more volume is present in the tube.

Step 5: Remove the syringe from the tube and add volume to the syringe. Connect the syringe and tube before unclamp tube 3 is done.

Step 6: Depress the syringe completely to extract out the volume present in the sampling tube into a test tube and clamp tube 3.

The 2nd part of the prac was used to make Luria-Bertani(LB) broth medium. Below are some pics as to how it was made by our group.

LB broth is used to seed the culture as well as fermentation media. The following steps are performed to prepare the 2L medium.

Step 1: 50g of pre-prepared mixture of Bacto-tryptone, yeast extract and sodium chloride was weighed.

Step 2: The mixture was dissolved in 2L of distilled water and autoclave at 121 degrees Celsius for 20 minutes.

E.coli was also streaked on an agar plate which was allowed for the growth before seeding into the fermenter.

That was all that was done on the first day =)

♥6:49 AM

-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