BPT blog

2010-01-19T09:08:00.000-08:00

Navigations are on top.

Sharing some Looooove! =D

2009-01-23T13:00:00.000-08:00

We sure had a blast of fun doing this practical! Not only did we scaled up the culture, we scaled up everything else. And I mean everything. We had tons of laughters and at the same time bringing our friendship and teamwork to greater heights. cheh! =P and yes, we gained knowledge too. hehehe.

Now thats quite an experience eh?

2009-01-23T08:18:00.000-08:00

2009-01-23T07:49:00.000-08:00

Preparation of equipment

The different parts of the fermenter and their functions were studied and summarized in the table below:

Preparation of media

1. 50 g of Luria Bertani (LB) medium was dissolved in 2L of water in a 2L bottle.
2. 100mL of the LB broth was then transferred in a 200mL shaker flask and the remaining 1900mL was transferred in the 2-L bioreactor.
3. The media was autoclaved at 121°C for 20 minutes.

4. When the broth has cooled down below 50°C, ampicillin was added to both the seed and fermentation media to a final concentration of 100µg/mL.
5. The media was then kept at 4°C until inoculation.

Preparation of culture (all the steps in this protocol were done aseptically)

1. pGLO transformed E. coli was retrieved from the -80°C freezer.
2. The cells were then streaked on a LB/Amp/Ara plate in the following manner showed below:

3. The plate was then incubated for 24 hours.
4. On the following day, several colonies formed on the plate were transferred to the flask containing 100mL LB medium with ampicillin.
5. The flask was then placed in a shaking incubator to incubate at 32°C for 24 hours.

Setting up and Monitoring Fermentation Process

1. After autoclaving, the 1900mL media that was remaining in the 2L bottle was transferred to the bioreactor.
2. The control parameters were then set as shown below:

3. A sample of 2mL was taken from the bioreactor before inoculation.
4. 100mL of the prepared seed culture was then inoculated into the bioreactor and fermentation was continued for 24 hours at the above conditions.

5. Samples of 2mL were taken from the bioreactor every hour for a period of 10 hours.

6. The optical density of the fractions were recorded at 600 nm

Isolation of GFP

· The cells were obtained by centrifugation at 10,000rpm for 5 minutes.
· The pellet was resuspended in 500µLof TE buffer of pH 7.5 and 2 drops of lysozyme were added.
· After 15 minutes, the tube was placed in liquid nitrogen till the contents were frozen.
· The tube was then thawed in warm water and the cycle of freezing and thawing was repeated for 2 times.
· Sonication was done on ice for 4 cycles of 25 seconds with 10 seconds rest in between sonication cycles.
· The contents of the tubes were spun down at 20 minutes at 10,000rpm
· The pellet and supernatant were separated and the pellet was resuspended using 400µL of TE buffer.

Purification of GFP

· 8 test tubes were labelled from 1-8 and one labelled blank.
· The blank was filled with 2.0mL of ammonium bicarbonate and the rest of the tubes were marked at the 2.0mL level.
· The column was carefully drained into a waste beaker.
· The cell free extract was transferred to the top of the gel bed
· The fractions were obtained by placing test tubes under the stopcock and filling each tube to a level of 2mL
· 50mM of ammonium bicarbonate were continuously added to the top of the column while taking the fractions.

· The optical density of the fractions were recorded again at 476 nm

2009-01-23T07:39:00.000-08:00

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.

2009-01-23T06:29:00.000-08:00

OBJECTIVES

Generating large quantities of a microbial product is not an easy task. We learnt that many considerations must be taken; type of bacteria, type of aeration, type of agitation, temperature, pH and monitoring processes amongst others. Care must be taken at every step as otherwise; the entire batch might be contaminated. Fermentation itself is quite useful to grow large quantities of cells and harvesting the product. Its ideas are simple, but putting it into practice is anything but easy.

Overall objective:
->To isolate green fluorescence protein (GFP) from E. coli

Experiment 1: Familiarisation with the Bioreactor and its Operation

->To get familiarized with the parts and components of microbial and mammalian bioreactors
->To be introduced to the basic operation procedure of a bioreactor

Experiment 2: Equipment, Media and Seed Culture Preparation

->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 3: Inoculation, Fermentation and Monitoring

->To carry out scale-up fermentation to increase yield of desired protein product (GFP in this case)
->To monitor cell growth and product formation through manual sampling and computer data logging

Experiment 4: Isolation and Purification of Product

->To carry out sonication to lyse cells and isolate cell content from cell debris
->To carry out gel permeation chromatography to purify the GFP

2009-01-23T04:21:00.000-08:00

2009-01-22T00:00:00.000-08:00

This is the record of the culture absorbance and fermenter parameters.

Hence, The Graph of cell growth !

&&&

Discussion of HISTORY PLOT !

In order for cells to proliferate and grow in high yields,
the following conditions must be present.

1. Dissolved oxygen (pO2

must be present to provide oxygen for the cells.

2. pH level

must be optimum.

3. Temperature

must remain constant.

4. Spargers and impeller

were required for aeration and stirring of the contents.

Hence, In this history plot,

pO2 value

- When cell are at the lag phase, the pO2 values have increased

as it was the period of adaption for the cells in a new environment.

- As the cells reaches exponential phase, the pO2 value have

hence, the demand of pO2 increased.

- After about 10hours, the pO2 values have started to increase.

It was due to the fact that cells are now in the stationery phase where; -
Rate of cell growth = Rate of cell death.
Hence the demand of pO2 was not high.

From the graph, we can observe that

the stirring speed and pO2 values are correlated to each other.

Stirring rate

- The stirring rate will be constant until the pO2 probe
detected that there was a decrease in the pO2 value.

- Level of pO2 increases = constant stirring rate

= no or less demand of oxygen from the cells.

- Level of pO2 decreases = Stirring rate increases

= demand of oxygen from the cells.

pH

- pH value was low initially as cells grow and produced

toxic metabolite substances which causes the pH to drop.

- As the pH probe detected the decrease in pH,

it will adjust the pH to the optimum value which is required.

Temperature

- Temperature was low at the start of the run

as cells were trying to adapt into the new environment.

- After about an hour later,

the temperature is remained constant throughout the whole run.

Theory

2009-01-21T00:45:00.000-08:00

Description and theory behind Green fluorescent protein

Aequorea Victoria is a type of jellyfish found off the cost of North America and can grow up to 5cm to 10cm. Tiny Aequorea Victoria are asexually budded of their hydroid colonies and feed on soft bodied prey which includes other types of jelly fish as well.

The interesting fact about this jelly fish is its ability to bioluminescent in the sea. They actually produce blue luminescent due to the protein aeqourin; however it appears as green to us; due to the fact that it is emitted through the molecule green fluorescent protein (GFP).

GFP was first discovered by the scientist Osamu Shimomura in the jellyfish Aequorea Victoria, and is a type of green fluorescent substance. The function of GFP is to transduce the blue fluorescent emitted by the jellyfish to green, through energy transfer. Since its discovery, GFP has been used in a wide range of application, for example the measurement of protein to protein interaction. Structure of GFP below:

The fluorophore of the GFP which is responsible for the fluorescent, originates from the internal Ser-Tyr-Gly sequence which has been modified to a 4-(p-hydroxybenzylidene) - imidazolidin-5-one structure. It is form by a series of events in an auto-catalytic process and no cofactors or enzymatic components are needed.

Due to the GFP being able to tolerate N- and C-terminal fusion to wide range proteins, it has been expressed in other animals such as the zebra fish and E.Coli. Furthermore, as GFP is non toxic and non invasive, it is being used as a marker for gene expression.

Transformation of E coli using pGLO gene

The pGLO vector contains several important bioreporter and biomarker sites, those of which include the Green Fluroscent Protein (GFP) site, ampicillin resistant site(Amp R) and arabinose site (araC)which codes for a protein that 'turns on' the GFP gene and allows it to fluoresce, hence the use of arabinose as feed in the experiment.

The GFP site allows the cell to fluoresce with help from the protein that is produced from the araC site.

The Amp R site codes for beta-lactamase. This allows the bacteria to break down ampicillin, hence confering ampicillin resistance to the bacteria.

A plasmid map of pGLO showing the three important sites- araC, GFP and Ampr

The GFP gene is first isolated and cut from jellyfish DNA with a restriction enzyme, and the gene is inserted into the plasmid ( depending on what kind of restriction enzyme is used) using ligase to stick the sticky ends to the plasmid.

The plasmid is then inserted inside the E coli cells, which hav

e had their cell membranes modified in order to take up the plasmids.

E coli cells are then grown on LB-Amp plates. The bacteria which do not acquire the plasmid die off as they get killed by the ampicillin present in the agar medium. Those bacteria that may have picked up the plasmid are able to glow in the dark as they possess the GFP gene.