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* Bioprocess Technology Practical Blog

this is : mb0604-4b.blogspot.com

This a blog used to record down and show all our procedures, photos, videos, results and discussions
for our Bioprocess Technology Practical sessions spanning over 5 days
to isolate the Green Fluorescence Protein from E.coli.
navigations on top. =)

Sunday, November 25, 2007 Our Learning Points

1:32:00 PM

Learning points about fermentation in general

· Fermentation is the oldest historical biotechnological processes method that many people had practiced during the past thousand of years.
· Fermentation is a process whereby an energy yielding anaerobic metabolic process in which microorganism convert nutrient to their products.
· In biotechnology, the term fermentation briefly referred to the growth of microorganism, under either aerobic or anaerobic conditions.
· http://biotech.about.com/od/glossary/g/Fermentation.htm

Wednesday, November 21, 2007 Results

10:58:00 PM

Answers to Questions

10:52:00 PM

Experiment 1
Answers to Questions

Q1:State the differences you observe between a microbial bioreactor and a mammalian cell bioreactor.
1. A microbial bioreactor has one rotameter while a mammalian cell bioreactor has four rotameters

2. A mammalian cell bioreactor does not have baffles while a microbial cell bioreactor has. This is so that to reduce the shear forces in the bioreactor as mammalian cells are fragile.

Q2: 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.
Exp 1:
It is to familiarize with the equipment of the fermentors and to have a rough idea what the fermentation process is about.

Exp 2:
Media for Seed-Culture and Scale-up Fermentation was prepared.
Bioreactor was prepared with pH probe, pO2 probe, foam, level probe, acid, base and antifoam installed to it.

Exp 3:
One loop of E.coli cells was transferred from the agar plate to the culture medium then, ampicilin is added to the culture.
The control parameters of the fermentor are set.
Culture (cells) is inoculated into the fermentor, and 10ml of sample is taken every hour after the culture is inoculated. 11 samples are taken (including the blank).

Exp 4:
Green fluorescent protein (GFP) is isolated.
Supernatant was taken out.
Isoenzyme was used to resuspend the supernatant so that to digest the bacteria cell wall.
The tube was freeze and thaw for 3 times to completely rupture the bacteria cell wall. Sonication by ultrasonic waves was used to complete the cell disruption.

Experiment 2
Answers to Questions

Q1 On media preparation
(a) Explain the purpose of each ingredient found in the LB media.
Bacto-tryptone: to provide the amino acids (peptones and peptides), carbon, energy and nutrients for bacterial growth.
Yeast extract: to provide a surplus of organic compounds useful for bacterial growth. Sodium Chloride is to provide sodium ions for transport and osmotic balance.
dH2O: solvent required for mixing the solution well to obtain a homogenous solution.
pH: optimal pH range that is suitable for the bacterial growth.

(b) What is the purpose of ampicilin?
Ampicilin is used to select desired genes which are usually the ampicilin-resistant genes that were already inserted into the bacterium.

(c) Why is ampicilin added only after autoclaving?
Ampicilin is sensitive to heat. Therefore, the addition of it after autoclaving will not alter the properties and effectiveness of it.

Q2
(a) What is meant by calibration of the pH probe?
Calibration of pH probe is to control the pH value of the fermentor by adding acid or base when the media is too acidic or basic.

(b) Why is hydrochloric acid not suitable as a correction agent for pH?
Hydrochloric acid is not suitable for correction agent for pH as it can fully dissociate in water so affecting the optimum composition of the fermentor. Also, HCl would be too corrosive to the bacteria cells and would damage the cells if used as a pH corrector. Therefore, H2SO4 is more commonly and ideally used to adjust the pH in fermentors.

(c) What is meant by polarization of the pO2 probe?
Using electromagnetic wave to control the pressure of oxygen of the pO2 probe.

(d) What is peristaltic pump?
A peristaltic pump is a type of positive displacement pump used for pumping a variety of fluids.

Q3
(a) What is the purpose of arabinose?
The purpose of arabinose is to provide a source of food and carbon for bacterial cells so that the bacterial cells can function properly.

(b) Describe the sterile techniques used in seed preparation.
Sterile techniques such as wearing gloves were used.

(c) Why do we perform step-wise scale-up instead of transferring directly to the fermentor?
This is to ensure that all contaminants are removed so that it does not affect the experiment.

Experiment 3
Answers to Questions

Q1: Explain the control philosophy for pH, temperature and dissolved oxygen as was used in the fermentation process.
pH: pH is measured by pH probe. In order for the pH to be at the desired level, constant monitoring have to be done. Acid is added when the media is too basic and alkali is added when the media is too acidic.
Temperature: Temperature is measured by the temperature probe. If temperature rises from the optimum 32 degrees Celsius, water will be pumped into it by the cooling jet.
Dissolved Oxygen: Dissolved Oxygen is measured by the dissolved oxygen probe. If dissolved oxygen is below the optimum level, air will be pumped into the fermentor by the sparger.

Q2: Describe the principle of the spectrophotometer which was used to determine the cell density (OD600). Why was 600 nm used?
Spectrophotometry is the quantitative study of electromagnetic spectra. It is because GFP emmission peak of more than 500nm.

Q3: 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 primary metabolite. It is harvested during the death phase.

Q4: 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.
The advantage of using a computer control system is that the need for manual control is eliminated. All the controlling mechanism is automated and the chores of maintaining the optimum fermenting conditions in the bioreactor is done by a computer system. Also, all the parameters for controlling the fermenting conditions in the bioreactor can be more efficiently controlled. From the history chart, it is shown that throughout the fermenting process, the temperature and pH levels were effectively maintained. On the whole, the graph fluctuated very slightly as the automated system detected the changes in temperature and pH and effectively carried out the corrective actions to bring the temperature and pH down to the optimum conditions.

Experiment 4
Answer to Question

Q1: Plot a graph of your A476 absorbance values (Y-axis) vs fraction number. Comment on your chromatogram.
According to the graph, it shows that the peak occur at fraction 2. The higher the concentration of the absorbing substance(GFP), the greater the amount of light absorbed. It is understandable that for fraction 1 and the last few fractions from 6-8, the GFP concentration is less. The absorbance for the fraction 1 is less as it takes some time for the sample to reach the other end of the chromatography column so the solution that is mostly collected is ammonium bicarbonate. And from fraction 2 onwards, the GFP will start to elute. Therefore, after few sample were collected, the GFP concentration will get lesser.

Q2: 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 of why not?

For size exclusion chromatography, the higher the molecular weight, the more likely the substance will get eluted first. This is because lower molecular weight proteins will be more likely diffuse into the gel resin slowing down the flow while higher molecular weight proteins will be less likely to diffuse into the gel resin therefore eluting first. For GFP which has a molecular weight of 27 000 kD and a protein with 50 000kD, the protein will be mote likely to elute before GFP as it has a larger molecular weight.

Further Questions:

1a )

What’s in the LB media ?

Bacto-tryptone, yeast extract, sodium chloride (NaCl), distilled/deionised water (dH2O).

Bacto-tryptone is a protein source for the grown of our E. coli.

NaCl provides sodium ions which E. coli utilises for transport and osmoregulation.

Water, to suspend the solids.

Yeast extract contains glutamate and vitamins, which are important in the processes of glycolysis, glucogenesis and the citric acid cycle. Vitamins act as catalysts and substrates in various cellular processes.

1b)
We add ampicillin with the intent for it to select only organisms with ampicillin resistance. Ampicillin competitively inhibits the transpeptidase produced by gram-positive bacteria to form cell walls, and thus kills them. Only bacteria with ampicllin resistance will flourish on the media, including our prized E. coli.

Plasmids inserted into the host cells coding for GFP were also coupled with beta-lactamase enzyme, causing the transformed cell to possess ampicillin resistance. Non-transformed cells would lack this property, and therefore would be killed. Other contaminants or microbes should they enter the media, will also be killed.

1c)
As ampicllin consists of proteins, autoclaving at high temperatures would result in denaturation and loss of activity.

2a)
The indication of the probe is adjusted, so that it would be of a greater degree of accuracy.

2b)
HCl will result in corrosion of certain parts of the fermenter, if it leaks out from the tubing. This would result in damage to the fermenter.

2c)
The pO2 electrode is comprised of a Teflon membrane chamber, with a platinum cathode, silver anode and saturated aqueous KCl. It works by the principles of electrolysis, by removing electrons from silver to produce silver ions. Electrons flow from the anode to the cathode where the oxygen undergoes reduction
A fixed voltage is applied between the cathode and the anode, so that the current flowing is proportionate to the oxygen dissolved.

2d)
A peristaltic pump is a displacement pump used for pumping a range of fluids, with contained within a flexible tube in a circular pump casing. Linear peristaltic pumps exist as well. A rotor with rollers compresses the flexible tube and fluid is forced through the tube by compression as it turns. This is highly similar to our biological system, hence the name.

3a)
Arabinose within agar allows the gene in the insert to be activated. Arabinose binds to the araC repressor, causing it to change and can no longer inhibit transcription of GFP gene.

3b)
Seed preparation was conducted in a more advanced fume hood as compared to normal labwork we previously experienced. Standard procedure for fume hood, UV treatment for 15 minutes and swabbed with ethanol. Hands were washed with soap, gloved donned and sprayed with ethanol before beginning work in the hood.
All media was swabbed with ethanol prior to entering the hood and inoculating loops were sterilised before use. The flask used was autoclaved, along with the media before E. coli was transferred to it.

3c)
Transferring directly to the fermenter would result in a longer lag phase, as the number of organisms would be too low, and thus have to take time to multiply to pass the lag phase. The steps we performed was to minimise the lag phase in the fermenter.

For reference:
http://en.wikipedia.org/wiki/Lysogeny_broth
http://en.wikipedia.org/wiki/Yeast_extract
http://www.eurasyp.org/public.levure.extrait.screen
http://en.wikipedia.org/wiki/Ampicillin

Monday, November 19, 2007 Last Day in Lab

1:31:00 PM

Procedure for Isolation and Purification of Product (page 16 to 20)

Isolation ( Stage 1 )

10 mL of culture broth was collected in a tube.
The cells were centrifuged at 10,000 rpm for 5 minutes.
Supernatant obtained was transferred into another tube
UV light was used to confirm the product present in the pellet.

The product inside the pellet would go through three different methods of cell disruption.

Method 1: Using Enzymes

Using a micropipettor, the pellent was resuspended in 500 µL of TE buffer (pH 7.5) till no visible clumps were seen.
Two drops of lysoyme was added into the resuspended cell pellet using 1mL plastic pipette and left untouched for 15 minutes.

Method 2: Freezing and Thawing

The tube was placed into liquid nitrogen until the contents are frozen.
The tube was then thawed using warm water.
Repeat the cycle of freezing and thawing for another two times.

Method 3: Sonication

4 cycles of 25 seconds with 10 seconds rest in between sonication cycles were carried out on ice.
The content was then centrifuged for 20 minutes at 10,000 rpm.
The pellet and the supernatant were then separated.
The pellet was then resuspended using 400 µL of TE buffer.
The product (GFP) now would be inside supernatant and to be purified.

Steve doing the sonification(he looks like a DJ :D )<br>

Purification (Stage 2)

Gel permeation or size exclusion chromatography was used to purify the extract obtained from stage

Procedure
1. Nine test tubes were prepared on a rack.
2. The tubes were then labeled 1-8 and another a blank.
3. 2.0ml of ammonium bicarbonate was added to the blank tube, and a marking is drawn onto the 2ml level as well as for the other 8 tubes, for easy reference when collecting fractions.
4. The buffer in the column was allowed to drain off into the waste beaker until the buffer touches the top of the gel bed.
5. The bottom of the column is then capped.
6. Cell-free extract was then added onto the gel bed gently and evenly by using a dropper.
7. The column was uncapped and the media allowed to flow through, the first fraction was collected. Make sure that the column does not dry up.
8. When the sample has entered the gel beads, 50mM ammonium bicarbonate will then be added to the column constantly to fill up the rest of the fraction until all the fraction have 2ml of sample elution.
9. The 8 samples were then taken to test for their absorbance value.

Analysis (Stage 3)

- Spectrophotometry

Absorbance readings were taken using a Spectrophotometer set at 476 nm . ( The wavelength at which GFP is strongly absorbs and gives out its usual florescence).

After the isolation and purification, the results were taken by using the spectrophotometer. The tubes were put into UV light and there is different degree of brightness to the tubes. This can confirm whether the reading is right as the brightest tube and the dimmest can be determined. This experiment shows that the bacteria cell wall is critical for the GFP product to be collected. Therefore steps like enzymes, freezing and thawing and sonification have to be done Lastly, gel permeation chromatography was used to purify the GFP product by passing it through 8 different tubes and result was taken after that.

Wednesday, November 14, 2007 Day 3

1:25:00 PM

Day 3, Bioprocess Labs, Q6.

Procedure for setting up and monitoring the fermentation process –

Scale-up fermentation:

Another day of bioprocess technology. Weary from the day’s onslaught of lectures we made our way to the lab, where we hoped we could skive off without being noticed.

The lab tech so kindly set up the scale up culture for us by transferring the seed culture into the fermenter via the peristaltic pump. With this, the E. coli cells can now be grown on a larger scale and thus produce the incredibly sought-after green fluorescent protein !

Continued from Day 1:

The medium broth was cooled to below 50°C and ampicillin was added to a final concentration of 100μg/mL and arabinose to a final concentration 0.2%.

The fermenter was innoculated with 100ml of seed culture, which translates to 5% of fermentation media volume.

The parameters our E. coli so desire:
Temperature: 32°C
pH: 7.5
Stirred speed: Min 10%, Max 90%, AUTO
pO2 setpoint: Set Point 20%, AUTO, Stir to CASC, Airflow to CASC
Airflow: Min 25%, Max 100%

This is where the Oxygen comes from

A 10ml blank sample was drawn before inoculation, and then subsequent samples every hour.

The blank sample was drawn by the lab tech, and we took turns to do our part on the hour. What exactly did we need to do, then ?

1 – Loosen the clamp on the inlet tube, before using the syringe to draw culture from the fermenter. The culture will flow into the sampling tube at the side of the fermenter.

2 – Tighten clamp back on the input.

3 – Loosen clamp on output tube, and remove syringe from exhaust. Draw air into syringe.

4 – Insert syringe to exhaust air filter outlet and push plunger.

5 – When the medium flows into the collecting tube, tighten the clamp.

And that was all we needed to do on our shifts ! (Pity the person who had the 6pm slot, everyone else was home free)
The fermentation broth was harvested after 24 hours in the fermenter, with 10ml of culture drawn into a sterile, disposable test tube, one tube per group.

The screen sample of bioreactor

Video for today's sample:

This is a graph generated by the monitering system showing the various parameters being measured and controlled.

Monday, November 12, 2007 Day 1 and 2

1:25:00 PM

Procedure for preparation of equipment, media and seed culture:

Media for Seed Culture and Scale up Fermentation –

Day 1 in the Bioprocess Technology labs, Q604.

Immediately after our briefing, our group 4-B started on some hands-on work immediately (albeit simple), which involved mixing 2 Litres of Luria-Bertani aka LB media. The 2 litres of LB media would go towards the seed culture and fermentation media later on.

Stirring

Measuring

The media preparation was deceptively simple, as we expected to be measuring various quantities of required components. Sadly, we were presented with the broth powder, which contained all the ingredients, and we were faced with the daunting task of... adding distilled water to make 2 litres of broth.

Pouring

Although relatively rusty with labwork, we managed this with ease.

End product

Day 2: transfering of media to shaker flask

100ml of the media was transferred into a 500ml shaker flask, and the remaining 1900ml went into the 2-litre bioreactor. Both the shaker flask and the bioreactor were set to autoclave for 20 minutes, at a temperature of 121°C. However we did not need to handle the autoclave or the addition of ampicillin as the lab technician took care of those tasks.

Shreaking of E.coli on agar plate

Ampicillin was added at to a final concentration of 100μg/ml in the shaker flask and fermentation media, after the broth was allowed to cool till an estimate of 50°C or below.

The media is now ready for inoculation, and is stored at 4°C until required.

Preparation of equipment – The Bioreactor:
Setting up the bioreactor from scratch seemed too complicated for us, as we were completely uninitiated and most were clueless as to where the controls were. However, the lab technician took care of the majority of the preparation, once again (he has our deep gratitude).

Essentially, he ran through all the control points of the bioreactor, and demonstrated to us the techniques we would need for drawing samples and other tasks once the fermentation process began.

I shall list the exact steps here:
1 – The pH electrode was calibrated with the use of standard buffer solution, and then install it, along with other probes for foam, pO2 and level. These went through the top plate of the reactor. Adjustments were made when necessary.

2 – Agent lines for acid, base and antifoam were added, linking the storage bottles and reactor. Storage bottles were checked.

3 – Exhaust condensers, air inlet, exhaust filters and manual sampler unit were installed, and the water jacket was checked to ensure that it was filled with water.

4 – All cables were disconnected except for the temperature probe, as it is autoclavable along with the bioreactor. All silicone tubings were clamped up except for the exhaust filter and female STT coupling of sampling unit. All filters and sockets were covered with aluminum foil to prevent moisture from condensing. When all this was done, the bioreactor was autoclaved at 121°C for 20 minutes. The pO2 probe was polarised for 6 hours and calibrated via aeration with nitrogen.

5 – Finally, the addition lines were connected to peristaltic pumps, and set to ‘Auto’ or ‘Manual’ control where required.

Preparation of Seed Culture:
After the overload of information regarding the bioreactor, we proceeded to prepare the seed culture.

pGLO transformed E. coli was taken from the -80°C freezer, and streaked on the LB/Amp/Ara plate and left to incubate for 24 hours.

Several colonies of pGLO transformed E.coli were transferred from an incubated plate to the flask containing 100ml LB medium with ampicillin.

The shaker flask was then placed in the shaking(uh, duh) incubator for 24 hours, at 32°C, which would later be used to inoculate the fermentor for scale up.

Video for today's procedures:

Getting to know parts of Bioreactor

1:30:00 AM

Getting to know Bioreactor:

Overall objectives

12:56:00 AM

Overall objective: To describe the entire fermentation process

Experiment No. 1: Familiarization with the Bioreactor and its Operation
Objective/s:
-To familiarize students with the parts and components of microbial and mammalian bioreactor
-To introduce the basic operation procedure of a bioreactor

Experiment No. 2: Equipment, Media and Seed Culture Preparation
Objective/s:
- 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

Experiment No. 3: Inoculation, Fermentation and Monitoring
Objective/s:
- To carry out scale-up fermentation process to increase the yield of desired protein product (Green florescent protein in this case).
- To monitor cell growth and product formation through manual sampling and
computer data logging

Experiment No. 4 Isolation and Purification of Product

Theory:
Green fluorescent protein (GFP) is a novel protein produced by Aequorea victoria which is a bioluminescent jellyfish. It comprises of 238 amino acids . It is often used in cell and molecular biology as a reporter of expression. This means that it binds to gene of interest to check whether the gene has been obtained or expressed in cell or organism population. The GFP has a unique can-like shape consisting of 11-strand β-barrel with a single alpha helical strand containing the fluorophore running through the center.

How to incorporate GFP gene into E.coli:

Vibrio fisheri and bacteria plasmid were first prepared. Restriction enzyme was used to cut it apart. Then they were put together to go through gene transformation by incorporating them into E.coli. Those genes that were not competent were washed away as Ampicilin was used to select those with GFP gene.