Theory behind green fluorescent protein
Recombination of plasmid is done to incorporate GFP into the E.coli bacteria. By using DNAse enzymes to cut out the desired gene from A. victoria. The same enzyme is then used to open the plasmid DNA from E.coli. DNA ligase is then used to ligate the desired gene into the plasmid vector of the E.coli. The plasmid vector is then inserted into the E.coli bacteria and a recombined E.coli is formed with the incorporated GFP gene.

Our purpose for this experiment is to isolate the green fluorescent protein. The green fluorescent protein can be found from a species of jellyfish, Aequorea Victoria. The green fluorescence is excited by blue light.

Its modified form, it can be used as biosensors and biomarkers to locate the gene of interest. I have here an interesting photo to share. It is done to show how diverse is genetic mutations.

The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins.

Learning points of Fermentation

Fermentation typically refers to the conversion of sugar to alcohol using yeast under anaerobic conditions. Fermentation usually implies that the action of the microorganisms is desirable, and the process is used to produce alcoholic beverages such as wine, beer, and cider. Fermentation is also employed in preservation to create lactic acid in sour foods such as pickled cucumbers, and yogurt.

There are different modes of fermentation namely, batch, fed-batch and continuous fermentation. Fermentation is also used much more broadly to refer to the bulk growth of microorganisms on some medium. No distinction is made between aerobic and anaerobic metabolism. This process is often used to produce or preserve food. The science of fermentation is known as zymology.

Overall objective – To scale up production of green fluorescent protein.

Experiment no.1 Familiarization with the bioreactor and its operation
To familiarize with the protocols for handling the bioreactor

Experiment no. 2 Media and Seed Culture Preparation
To prepare the bioreactor
To prepare for seeding the culture
To scale-up fermentation of culture

Experiment no. 3 Inoculation, fermentation and monitoring
To increase yield of desired protein product (green fluorescent protein)
To monitor cell growth and product formation by manual sampling and measuring absorbance and computer data logging of environment factors in the fermentor

Experiment no. 4 Isolation and purification of product
To harvest the product from the fermentor
To isolate and purify the product GFP(Green fluorescent protein) from the harvest

We leant more about the bioreactor and its components.

Wrap equipments with aluminum foil to prevent condensation after autoclave.
Media for Seed-Culture and Scale-up Fermentation
1. 2.0litre of distilled water was added to 50g of LB (Luria-Bertani Medium)
2. Once media was prepared, it was autoclaved for 20 minutes.
3. Ampicillin and arabinose was added after the media was cooled to below 50°C.
4. The pH electrode was calibrated using standard buffer solution.
5. The pH probe, pO2 probe, foam, level probe, exhaust condensers, air inlet and exhaust filters were installed.
6. Escherichia coli containing the Green Fluorescent Protein were streaked onto an LB plate.

Answers to further questions:

Differences between microbial bioreactor and mammalian cell bioreactor:

1. E.coli colonies were seen on the plate.
2. The colonies were transferred into a shaker flask. (100ml of LB broth and Ampicillin).
3. Arabinose was added into the culture medium.
4. Parameters of the culture medium were adjusted for optimum cell growth.

Answers to further questions:

On media preparation:

Peptide and peptone(bacto-tryptone) - provide essential amino acid to growing bacteria.

Na+ ions (NaCl) - for transportation and maintining osmotic balance

Trace elements (Nitrogen, Sulphur, Magnesium) - provide a plethora of organism component helpful for bacteria growth.

Purpose of Ampicilin is to inhibits the growth of other bacteria thus preventing contamination

It is added only after autoclaving because Ampicilin will be inacivated at high temperature.

On equipment media:

Calibration of pH probe is to maintain the pH of media using buffers.

Hydrochloric is a strong acid. It dissociates in water affecting the optimum composition of the fermenter.

A peristaltic pump is a type of positive displacement pump used for pumping a variety of fluids. The fluids are pumped, just like the wavy-like motions of the oesophagus.

On seed preparation:

Arabinose is to provide the essential nutrients and carbon for bacterial to maintain the functions of the bacteria cells.

Gloves are worn when transferring E.coli into the flask containing LB medium, thus preventing contamination of any other bacteria found on hands. pGLO transformed E. coli is kept in the freezer to prevent any bacteria from multiplying. Bacteria cannot survive or multiply under extreme low temperature.

Step-wise scale-up is performed instead of transferring directly into the fermenter to ensure all the contaminants in the fermenter are being removed thus preventing contamination.

The fermenter was inoculated with 100ml of seed culture and 10 ml of sample was taken out every hour. (8am to 6pm).
How to extract from the seed culture:
i) The clamp between the fermentor and the sampling tube was first loosened.
ii) The syringe was drawn back to extract the sample into the sampling tube.
iii) The syringe was pushed back. This would eject the sample that was within the tube, back into the bioreactor.
iv) The clamp between the fermentor and the sampling tube was tightened.
v) The syringe is taken off the filter. It was pulled back and fitted back into the filter.
vi) The clamp at the outlet was loosened.
vii) The syringe was pushed to empty the content in the sampling tube.
viii) The outlet was tightened with the clamp.
ix) The sample is obtained.

Answers to further questions:

pH, temperature and dissolved oxygen are conditions that are required for cell growth. All these conditions can be controlled for optimum cell growth. The optimum pH level that the bacterial cells can grow is between 6.5 to 7.0 cells will denature if conditions are not favourable. Dissolved oxygen is important but excessive oxygen will cause damage to the cells as free radicals are produced. Temperature is an extremely sensitive condition for cell growth. Temperature above 40°C will cause the bacterial cell to denature.

600nm is used because that is the wavelength that cells absorbs the most. Light is emitted out from the source into a prism so that the ray is scattered (different coloured rays). The rays continue to shine through a cuvette containing the sample. Depending on the sample inside the cuvette, different wavelengths are emitted and recorded by a detector. The detector will convert the light into OD (optical density) readings.

Environmental control of the fermentor can be controlled with more accuracy. Probes in the fermentor is able to detect minute changes in pH, temperature, dissolved oxygen and foam levels and the computer control system can adjust to it appropriately. From the history chart, it shows the stability of cell growth.

Green Fluorescent Protein is a primary metabolite. It is produced during the primary growth, lag and log phase. The products are harvested in the stationary phase.

Culture harvest.
Lab technician showed us the overall graph plotted by the computer. The graph comes with 4 conditions. pH, O2 reading, stirrer speed and temperature.

Isolation
In order to obtain Green Fluorescent Protein (GFP) which is an intracellular product, the bacteria cells need to be lysed first to release the protein.

10mL of the culture broth was collected and centrifuged at 10,000rpm for 5 minutes to obtain cells which will form a pellet at the bottom of the tube. Poured the supernatant into another tube and observed both tubes under UV light to confirm the product.

Three methods used to disrupt the cells:
Using enzymes
Freezing and thawing
Sonication

Stage 1
Green fluorescent protein is an intracellular product, hence the bacteria cells needed to lysed first to release the protein. Three methods of cell disruption were performed on the bacterial cells.
1. 10ml of culture broth was collected in a tube.
2. Cells were obtained by centrifuging at 10,000rpm for 5 minutes. Pellet contained the product. 3. Pellet was viewed under UV light.

Method 1 – Using Enzymes
a. Pellet was re-suspended in 500µl of TE buffer of pH 7.5 using a micropipettor.
b. 2 drops of lysozyme was added to re-suspend the pellet.

Method 2 – Freezing and Thawing
a. Tube was dipped in liquid nitrogen to freeze the suspension.
b. Cell rupturing was completed by quickly putting the tube into hot water.
c. Cell wall expands (when frozen) and contract (when thawed).

Method 3 – Sonication
a. Cell disruption is completed by the process of sonication where ultrasonic waves (of higher frequency than sound) cause the bacteria cell wall to implode under the vibration pressure.
b. Sonication on ice for 4 cycles of 25 seconds with 10 seconds of rest in between.
c. The product is in the supernatant.

The cell disruption is completed by the process of sonication where ultrasonic waves cause the bacteria cell wall to implode under the vibrational pressure.

Stage 2 - Purification
Extract from stage 1 was purified by Gel Permeability Chromatography or Size Exclusion Chromatography.

This method of purification uses a column of a polymer gel resins (Sephadex G75). The resins contain very small pores in which molecules which are small enough can diffuse within.Hence, when the extract is poured into the column, the larger molecules will spend more time interacting and diffusing into the pores of the gel resins. This achieves separation of the different molecules by size.

1)Label 8 test tubes and a blank
2)Filled the blank with 2mL of ammonium bicarbonate
3)Added cell free extract into the column of gel
4)Collected buffer and extract to the 2mL mark of the test tube while adding ammonium bicarbonate
5)Exposed the tubes to UV light and measured absorbance

Stage 3 – Analysis

Take absorbance readings using spectrophotometer set at 476nm.


Results for Products formed

Discussions

In this experiment, both of the graphs show us that during the 1st hr of putting in the cells to the bioreactor, the cells are at a lag phase where they are trying to adapt to the new environment which they are in. As shown in fig 1 (calculation), the OD reading of the cells are really low, which reads at 0.001.The oxygen level used was the lowest almost the whole graph seen. The pH level during the lag phrase was stable, since the cells have not started consuming any substrate and oxygen, and no waste have been produced yet.

After the 2nd hour, the cells have started to adapt to the new environment given to it. Therefore, the using of the substrate & the oxygen level start to increase which was shown in the graph Fig.2 the decreasing line of PO2 plotted by the computer showing that the amount of the oxygen level found in the bioreactor was not sufficient and air was pumped into the reactor for the production of new cells. The Abs reading was the highest. Oxygen is also one of the important factors that will affect cell growth, if the amount of oxygen needed to produce new cells is not enough, cells will not grow properly.
As cells start to multiply and use substrate, the pH inside the bioreactor will drop or increase due to the by-products of the cells and will not be at optimum for cell growth, so the reactor will automatically add in acid or base when it detects that the pH level in the reactor changes.
Foaming is also another factor with will affect the rate of the cells growth, as oxygen is sparged into the bioreactor as bubbles through the sparger & the impeller are moving at a high speed (proportional to the amount of oxygen level in the bioreactor) to provide enough oxygen to the cells, therefore foam may form, foam may cause cell to lyses when the bubbles pop. The media will be reduced by creating foam too and anti-foam is added to reduce levels of foam when the foam probe detects foam present. Most bioreactors will have baffles at the side to create a more uniform mixing by interrupting the whirlpool effect caused by stirring from the impeller.
This is called the exponential phase as it is the phase where the largest amount of oxygen level consumed by the cells for cell growth, more foaming will occur during this phase & the impeller tend to stir at a higher rate shown from the Fig 2.

During the stationary phrase after 6hr, the cell growths are already to the maximum, shown by Fig 1 (calculation) the log phase is 0 & the fig 2 of utility of the oxygen, anti-foam & the stirring speed of the impeller are more stable, no infrequently of up/down, which the cell growth are slowly going down nearly coming to the death phase.


Results for Isolation and purification of product

Absorbance reading set at 476nm.

Discussions
The graph shows the absorbance value of the GFP substance. The graph shows that fraction 3 has the highest OD reading as compared to the rest. This shows that fraction 3 has the highest concentration of GFP. When shown under UV light fraction 3 presented the highest glow intensity then fraction 4 and 6. The points after fraction 4 are off; this shows that the absorbance readings are off the decreasing scale. The points are fluctuating. Theoretically, the amount of GFP should decrease as more buffer are added through the column and eluting the GFP. But at fraction 6 in the graph the absorbance value is slightly higher then fraction 5. Most amount of GFP being eluted is in fraction 3 followed by 4 in the graph. The absorbance reading of fraction 9 in the graph is almost at 0.000; this concludes that most of the GFP has been eluted from the column.

Answers to further questions:

Sephadex G-75 contains small pore resins which do not allow large molecules to interact with. Large molecules like proteins can’t interact with Sephadex G-75, thus, a protein of Mr 50,000KD will elute first before the GFP with Mr of 27,000. Since protein are larger then GFP, it will pass through the column faster then the GFP and being eluted first. The GFP will then take its time to slowly diffuse in and interacting with the pores.