Experiment 4 (Day 5): Further Questions

The answers below adress the questions in our practical manual.

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

Refer to the discussion section in Experiment 4 (Day 5).

2) GFP has Mr (molecular weight) around 27, 000 kD. Though we were unable to see them, the cell 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 with a Mr of 50, 000 kD would elute in a fraction before GFP, this is because it is bigger than GFP. Therefore larger protein, which fit in fewer pores, takes a more direct path through the column and elute first. The smaller GFP are slower to elute as they meander in and out of pores spending less time in the liquid mobile phase of the column.

Experiment 4 (Day 5)

Isolation and Purification of Product

Objectives

1. To isolate the desired product using enzymes, freezing & thawing and sonication to obtain an extract needed for purification.
2. To purify the product via gel permeation chromatography and separate impurities by their different sizes.
3. To analyze the wavelength at which the green fluorescent protein strongly absorbs and give out its usual absorbance by taking absorbance readings using a spectrophotometer set at 476nm.

In this experiment, the Green Fluorescent Protein (GFP) is isolated from the E. coli in the sample by cell disruption techniques (using enzymes, freezing and thawing and sonication) and then purified using gel permeation (a.k.a. size exclusion chromatography). 2 tubes of 10ml broth culture were made to under go the 3 cell disruptions and the purification stages.

Stage 1 – Isolation

The procedure for obtaining a cell pellet from the culture broth is shown below.

1) Firstly, we had to obtain 10ml of culture broth in two test-tube for the rest of the experiments.

2) In order to separate the cells from the liquid broth, we have to centrifuge the culture broth at 10,000 rpm for 5 minutes.

3) A pellet would be formed at the bottom of the tube as the cells are dense whereas the liquid broth is less dense and forms the supernatant.

4) The supernatant was then poured into another tube and both tubes were observed under UV light to confirm that the GFPs are inside the pellet.

The procedures for each of the 3 cell disruption methods are shown below.

Enzymatic Method

1) Firstly, the pellet was resuspended in 500µl of TE buffer of pH 7.5 with a micropipette until there were no visible clumps.

2) Next, 2 drops of lysozyme were added to the resuspended pellet whereby this will initiate the enzymatic digestion of the bacteria cell wall.

3) The enzyme was then allowed to act on the resuspended pellet for 15 minutes.

Freezing and Thawing Method

1) After 15 minutes, the tube was placed in liquid nitrogen until the contents are frozen.

2) Subsequently, the tube was thawed in warm water.

3) The cycle of freezing and thawing was repeated for another 2 times to complete the rupturing of the bacteria cell wall.

4) Freezing and thawing add mechanical stress to the cell wall as the cell water content expands (when frozen) and contracts (when thawed) causing it to rupture.

Sonication Method

1) The cell disruption is completed by the process of Sonication where ultrasonic waves (of higher frequency than sound) are used to implode the bacteria cell wall under the pressure of ultrasonic waves’ vibration.

2) Sonication is done on ice for 4 cycles of 25 seconds with 10 seconds rest in between Sonication cycles so as to prevent denaturation of the GFP protein.

3) Next, the contents of the tube were centrifuged after cell disruption for 20 minutes at 10,000 rpm.

4) The pellet was separated out of the supernatant as done previously during method 1.

5) The product (GFP) is now in the supernatant and pressence of GFP can be seen by observing the tubes under UV light.

Stage 2 – Purification

Purification to obtain the GFP protein was done on the supernatant obtained in the isolation steps using size exclusion chromatography. This method of chromatography uses a colum of polymer gel resins (Sephadex G75) which contains small pores as the stationary phase. When the extract flows through the column, smaller molecules interact with the pores of the resin and elutes slower. However, the larger molecules which are too large to interact with the pores will flow through the column faster.

The procedure below shows how this is done.

1) Firstly, eight test tubes were labeled from ‘1’ to ‘8’ and another test tube was labeled as ‘blank’.

2) The blank was then filled with 2.0ml of ammonium bicarbonate. Using this tube as a rough guide, the rest of the test-tubes were marked at the 2.0ml level.

3) The column was next allowed to drain into a waste beaker until the buffer is just above the top of the gel bed.

4) Subsequently, our cell free extract obtained from the isolation steps was transferred into the top of the gel bed by swirling around the inner edge of the column.

5) Next, the stopcock was opened and the sample was allowed to flow into the gel bed and the eluting buffer was collected in the first tube. While the eluant (buffer) flows into the tube, more eluting buffer (ammonium bicarbonate) was added to the top of the column to ensure that it does not run dry.

6) Once the sample has filled the tube to the 2cm mark, the tube was swapped with the next. This was done for the remaining tubes while ensuring that the gel bead does not run dry.

Note: The column was washed with 50ml of ammonium bicarbonate after the experiment so that it could be reused.

Stage 3 – Analysis

After the purification, the solutions from each of the 8 tubes and the blank were transferred to cuvettes and their absorbance readings were taken using a spectrophotometer set at 476nm. This was done for the other set of tubes obtained from the other set cell extract. 476nm is the wavelength at which GFP strongly absorbs light and gives off fluorescence.

The results showing the absorbance readings are shown below.

The graphs show the absorbance readings against their respective fractions of the 2 samples.

Click image to enlarge

Discussion

For discussion purposes, only sample number 2’s graph is used.

Since GFP absorbs light at the wavelength of 476nm, the absorbance obtained is indicative to the amount of GFP present. Hence from observing the absorbance readings and the graph fraction 2 has the highest amount of GFP followed by fraction 1 and fraction 3. Fractions 4 to 8 possess no GFP due to the 0.000 absorbance.

From the 1st fraction to the 2nd fraction, the absorbance increased from 0.385 to 2.745 and from the 2nd fraction to the 3rd fraction it then decreased to 0.054. Hence it can be deduced that the GFP probably had started eluting from the 1st fraction and stopped eluting at the 3rd fraction as a peak is observed spanning the from the 1st to the 3rd fraction as seen from the graph. The 1st fraction is lower in absorbance as the GFP protein requires sometime to elute through the column and would contain a larger amount of ammonium bicarbonate. The 2nd fraction is the highest as this is where most of the GFP is eluted from the column. The 3rd fraction has a low absorbance as almost all the GFP have eluted and only the remaining amounts are collected here.

Hence to obtain the purified GFP product, fractions 1 to 3 should be used and pooled.

Note: There are additional questions to help in better understanding of this experiment. The question and answers are located in the next post.

Experiment 3 (day 3): Further Questions

The answers below adress the questions from our practical manual.

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

pH – As the bacteria cultured in the fermenter grow, reproduce and carry out metabolic activities, waste materials will be produced. Some of these waste materials that accumulate in the culture broth are acidic and will cause the pH of the broth to decrease. If the pH is allowed to decrease till toxic levels, the bacterial growth will stop or the bacterial cells in the fermenter might die. Depending on the strain and type of the microbe being cultured the waste products might increase or decrease the pH.

Hence to prevent extreme pH deviations from affecting cell growth and survival, acid (for high pH) or bases (for low pH) are pumped into the fermenter via peristaltic pumps to correct the pH back to optimum levels.

Temperature – There is an optimum temperature for the growth of the cultured microbe and the yield of the product it produces. Hence the temperature of the fermenter must be kept at optimum levels for optimum growth and production of the product of interest (GFP). This is done by the cooling jacket of the fermenter where hot or cold water can be pumped into to correct deviations from the optimum temperature.

Increase in temperature could be due to factors such as microbial respiration, movement of the impeller. Decrease in temperature could be due to factors such as cooling from the surroundings (air-conditioning) or endothermic reactions occuring within the cells.

Dissolved Oxygen – The culture requires an optimum amount of dissolved oxygen to provide a sufficient oxygen supply for cellular respiration. If too little dissolved oxygen is present, the growth rate of the culture might slow down. However, if the dissolved oxygen is too high it may become toxic to the cells as high levels of oxygen might generate reactive oxygen species that could damage the cultured cells’ DNA causing cell death.

To regulate the dissolved oxygen levels in our fermenter, the stirrer speed is varied. If the dissolved oxygen is too low, the stirrer speed will increase to break the air bubbles sparged in by the sparger into smaller bubbles. The smaller bubbles provide a larger interfacial area for oxygen to diffuse out of the bubbles and reach the cells in the culture. The reverse happens for high dissolved oxygen levels.

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

The spectrophotometer can be used to detect substances that are able to absorb light which in this case are cells by emitting light at a particular wavelength (600nm in this case). This emitted light passes through a cuvette which holds the sample and are absorbed by cells found within the sample. The light which is not absorbed is transmitted across the cuvette into the detector which detects the amount of this transmitted light. The reading is translated into absorbance which is the amount of light absorbed by the sample.

By using Beer-Lambert’s law,

A = ε x l x c

Where,
A is the absorbance,
ε is the extinction coefficient (ml mg-1 cm-1)
l is the path length of light (cm),
c is the concentration (mg/ml),
the cell density can be calculated.

The cell density was measured by using light at wavelength 600nm as it is the best wavelength at which bacterial cells of a certain range of sizes can absorb light. If the wavelength is small, the light might be absorbed by things like proteins and DNA and the spectrophotometer will detect them too. Moreover, if the wavelength is too large, light will not be absorbed by the cells.

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 which is produced during the stationary phase. Since GFP is produced at the stationary phase, the product should be harvested at the stationary phase just before the start of the death phase. The product was actually harvested at the stationary phase.

3) 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 advantages of using a computer control system are:

• Ability to do data logging, data analysis and process control
• Allow for automatic adjustment of variables to correct deviations from optimum levels.
• Highly efficient in correcting deviations
• Allow the use of lesser manpower as no extra labour is required to constantly monitor and make corrects to deviations from optimum levels of variables.

Please see Experiment 3 (Day 3) for the history chart.

Computer control in our fermentation is relatively efficient. As soon as a deviation from the set point is detected, the respective corrective action is immediately take. For instance, when the dissolved oxygen decreases below the set point, the pO2 probe will detect this change and relay this information to the computer. The computer will then automatically calculate the degree to which the stirrer speed is to be increased to increase the dissolved oxygen levels in the fermenter. It is these kinds of controls that help keep the media and growth conditions as favourable as possible for cell growth and which makes computer control effective.

Experiment 3 (Day 3): Growth Table Calculations

Since the equation have been simplified from to {Log [A / (A0) ] },

Log ( X / X0) = {Log [ A / (A0) ] }

Therefore, to calculate Log ( X / X0) it is being replaced with {Log [ A / (A0) ] }.



For sample 1, absorbance is 0.431 while A0 is 0.308

{Log [ A / (A0) ] } = {Log [ 0.431 / (0.308) ] }
= 0.146

For sample 2, absorbance is 0.397 while A0 is 0.308

{Log [ A / (A0) ] } = {Log [ 0.387 / (0.308) ] }
= 0.099

For sample 3, absorbance is 0.408 while A0 is 0.308

{Log [ A / (A0) ] } = {Log [ 0.408 / (0.308) ] }
= 0.122

For sample 4, absorbance is 0.477 while A0 is 0.308

{Log [ A / (A0) ] } = {Log [ 0.477 / (0.308) ] }
= 0.162

For sample 5, absorbance is 0.483 while A0 is 0.308

{Log [ A / (A0) ] } = {Log [ 0.483 / (0.308) ] }
= 0.195

For sample 6, absorbance is 0.427 while A0 is 0.308

{Log [ A / (A0) ] } = {Log [ 0.427 / (0.308) ] }
= 0.142

For sample 7, absorbance is 0.410 while A0 is 0.308

{Log [ A / (A0) ] } = {Log [ 0.410 / (0.308) ] }
= 0.124

For sample 8, absorbance is 0.507 while A0 is 0.308

{Log [ A / (A0) ] } = {Log [ 0.507 / (0.308) ] }
= 0.216

For sample 9, absorbance is 0.526 while A0 is 0.308

{Log [ A / (A0) ] } = {Log [ 0.526 / (0.308) ] }
= 0.232

For sample 10, absorbance is 0.532 while A0 is 0.308

{Log [ A / (A0) ] } = {Log [ 0.532 / (0.308) ] }
= 0.237

Experiment 3 (Day 3)

Inoculation, Fermentation and Monitoring

Objectives

1. To perform scale-up fermentation process to increase the yield of green fluorescent protein by monitoring and altering the fermentation conditions.
2. To monitor cell growth and product formation through manual sampling for every hour over a period of 10 hours and interpretation of the different phases via computer data logging.

Part I-Procedure for Setting up the Fermentation Process

The steps below outline what was done to prepare the fermenter to start the fermentation process.

1) Firstly, the medium broth in the fermenter had ampicillin added to a final concentration of 100μg/ml and arabinose to a final concentration of 0.2% after it has cooled to below 50oC after autoclaving.

2) The control parameters were set as shown below.

• pH ----------------------- 7.5
• Stirring Speed ----------- Min 0%, Max 90%, Control set to “AUTO”
• pO2 Set Point ------------ Set point 20%, Control set to “AUTO”
• Air Flow ----------------- Min 25%, Max 100%
• Set “Stir” and “AIRFLOW” to “CASC"

Temperature which is supposed to be set at 32oC is not done in this case as the temperature control module of the fermenter was out of order. Hence fermentation was carried out at ambient temperature (room temperature).

3) 100ml of the seed culture (5% of fermenter media volume) was then inoculated into the fermenter using a pump.
4) Fermentation was then allowed to continue for 24hours.

Part II-Procedure for Monitoring the Fermentation Process

The steps below outline what was done to monitor the bacterial growth and various parameters throughout the fermentation process.

1) The parameters such as pH, stirring speed and airflow were monitored by the computer and recorded in a graph. If any of the parameters deviate past the optimum range, automated corrective actions will be carried out by the computer and the various pumps.

2) For every hour within the first 10 hours from the start of the fermentation, samples were drawn from the fermenter and kept in falcon tubes. The steps taken to draw the samples are shown below.

1. Ensure the tubing after sampling tube (tubing placed with one end in 70% ethanol) is clamped tightly.
2. Open the clamp on the tubing before the sampling tube (tubing connected with one end to the fermenter).
3. Draw the culture to approximately 1/3 of the sampling tube by pulling the syringe.
4. Push back the syringe to force the media back into the fermenter (The media that remains in the tubing before the sampling tube.) This step is done so that the next sample we draw will contain the culture in the fermenter and not the one left in the sample drawing tube.
5. Next, clamp the tubing before the sampling tube back tightly.
6. Take out the syringe and pull to fill it with air then place it back.
7. Unclamp the sampling tube and push the syringe to collect the sample in a falcon tube
8. Clamp the tube back and repeat these steps for subsequent sample drawing.

3) The optical density readings of the samples collected during the first 10 hours was taken using a spectrophotometer set at 600nm on another day and analysed.
4) The graph of the parameters through the course of the whole fermentation was also analysed.

The video below shows how samples were taken from the fermenter.

Part III-Monitoring Cell Growth

Results

The results of the Absorbance obtained from the OD600 readings are shown in the table below:

The absorbance of the samples taken within the 10 hour period was measured at 600nm.

Using the formula:

A = ε × C × l

Where A = measured absorbance, 600nm
ε = extinction coefficient
C = concentration of the protein (in mg/mL)
l = length of light path through solution, 1cm cuvette is used

Derived using the equation above,
[Equation 2]: C = A / (ε × l)
[Equation 1]: C0 = A / (ε × l)

Substituting 1 into 2,

Log ( X / X0) = Log ( C / C0)
= {Log [ A/ (ε × l) ] / [ A0 / (ε × l)] }
= Log [ A / A0 ]

Hence,
Log ( X / X0) = Log [ A / (A0) ]

*The calculations for the table are shown here.

The graph of Log (X/X0) against time,

Discussion

The graph above shows log(X/Xo) against time where log(X/Xo) represents the concentration of the E. coli cells in the fermenter.

From inoculation at 0 hours till the 1hour into the fermentation, there was a sharp increase in the cell concentration before falling in concentration from the 1st hour to the 2nd hour of fermentation. This sharp increase could be due to the cells entering the exponential phase almost directly after inoculation and growing at an exponential rate while the decrease from the 1st to the 2nd hour of fermentation could be due to cell death arising from changes in the media conditions (eg. pH was not well maintained). However, this is unlikely to be the case since the media conditions are well regulated by the computer and an absence of a lag phase is highly unlikely. Hence, the likely explanation for this could be that the sample taken at the 1st hour of fermentation was done using incorrect sampling techniques or the OD reading was not taken properly.

Due to this discrepancy, we will take that the portion from 0 to 2 hours, the curve slopes upwards gently to indicate the presence of a lag phase were the cells would be adjusting to the new environment before sloping further to join the original graph at 3rd hour to indicate the start of the exponential phase where the cells would be increasing in numbers at an exponential rate.

From the 3rd hour onwards till the 5th hour, there is an increase in cell concentration at a relatively high rate. This indicates that within this period, the cells are in the exponential phase and growing in numbers at a high rate.

From the 5th to the 7th hour, the cell concentration dropped before having a sharp increase again till the 8th hour. The decrease in cell concentration could be due to cell death as the E. coli cells proceed into the death phase. This could have arisen due to accumulation of toxic waste products or depletion of cellular reserves of energy. The sharp increase in cell concentration on the 7th hour could be due to cell death in the 5th to 7th hour. When the cells die some of them will lyse releasing their intracellular contents which can be used by other live cells as nutrients. Due to this increase in nutrients due to cell lysis, the living E. coli cells will use these nutrients to grow and enter a secondary growth phase. It is the sharp increase in the 7th to 8th hour that the secondary growth phase’s exponential phase occurs as seen by the large sudden increase in cell concentration.

From the 8th to 10th hour there is a very slow increase in cell concentration as the rate of increase of cell concentration begins to slow down. This period is most likely the beginning stationary phase of the secondary growth curve where the cell growth rate is almost equal to the cell death rate and is usually caused by the exhaustion of a critical nutrient or accumulation of toxic waste products. It is at this phase where maintenance of the cells occurs, though there is increase in cell concentration, metabolism still occurs. Secondary metabolites such as our protein of interest, GFP is produced at this stage and accumulated intracellularly.

Part IV-Part Monitoring Parameters

Results

The graphs below shows the data obtained from the computer regarding the various parameters that were controlled by it over the course of the fermentation. This graph was not that of our experiment but that of a different class from previous years’.

Click Image to Enlarge

Discussion

The graph above shows the history plot of the different variables (pH, pO2, stirring speed and temperature) measured by probes and plotted by the computer during the course of the fermentation.

pH

The pH was held rather constant throughout the course of the fermentation beginning at a pH of 7.40 and held constant at 7.40 until 6.25 hours where it increased to pH of 7.55 and held constant at 7.55 for the rest of the fermentation. The small increase could be due to the fact that the bacteria might be growing exponentially at that portion and probably is in the middle of its exponential growth phase, releasing an increased amount of metabolic waste products that could have caused a slight increase in pH. The region before was maintained at a lower pH level of 7.40 as the bacteria could be in the lag or early exponential phase and hence not produce as much metabolic waste to significantly affect the pH as much.

The pH was kept rather constant throughout the fermentation run is it was controlled and monitored by the computer which will correct any deviation from optimum pH if they occur by adding base to increase the pH if it falls below optimum or by adding acid to decrease the pH if it increases above optimum.

pO2

The dissolved oxygen drops initially from about 80% to about 15% during the first 3.75 hours of fermentation. This drop is due to the cells utilising the oxygen for their metabolism. Even though the cells are usually in the lag or very early exponential phase during this time and the cells are not increasing much in cell concentration, metabolism still occurs to allow the cells to survive hence the drop in pO2 levels are seen. The pO2 levels are not kept steady and constant here by the computer as the set point is set at 20% which means it will start to correct deviations only if it falls below this set point.

After 3.75 hours many spikes are seen till the approximately 9th hour of fermentation, this is probably when the cells are undergoing their exponential growth phase and are experiencing their highest growth rate and metabolism and is where oxygen utilization is at its highest. Hence as the dissolved oxygen oxygen is rapidly utilised and falls below the set point, the computer actively corrects this deviation by increasing the dissolved oxygen by increasing the stirrer speed. This process occurs many times causing numerous spikes to appear on the graph.

From the 9th hour of fermentation till approximately 18.75 hours the dissolved oxygen level remained relatively constant at 20%. This is probably when the cells are within the stationary phase to the death phase where the cells are no longer growing or dying and is a period of maintenance, hence there is minimal usage of oxygen for metabolism hence not much dissolved oxygen is used. Hence the graph shows an almost linear line without spikes as the computer is not correcting any deviations of pO2.

From the 18.75 hours till 21.5 hours many spikes are seen as the cells undergo a secondary growth curve and begin to enter the exponential phase of that curve. The reasons behind the spikes are the same of that from 3.75 – 9 hours.

After the 21.5 hours the pO2 levels begin to increase. This could be due to cells undergoing death phase or harvesting when the oxygen utilisation by the cells decreases and rate of the surrounding oxygen dissolving in the media surpasses the oxygen utilisation of the cells causing an increase of pO2 beyond the set point.

Stirrer speed

The stirrer speed corresponds directly to the dissolved oxygen level where it increases when the dissolved oxygen is low and decreases when the dissolved oxygen is high and hence matches the pO¬2 graph. It remains constant during the first hours of fermentation before rising and producing spikes, followed by a decrease, a constant level of stirring, an increase followed by spikes and finally a decrease.

Stirrer speed affects the dissolved oxygen level as by increasing the stirrer speed, it breaks up the bubbles from the sparger into smaller bubbles which have larger interfacial surface for more efficient diffusion of oxygen. This increased diffusion increase the dissolved oxygen levels in the media.

Notice that the stirrer speed never falls below 200rpm as a certain level of agitation is required to ensure that the cells do not settle to the bottom of the fermenter.

Temperature

The temperature in the fermenter was kept constant at 32oC throughout the whole fermentation run. The small spikes occur due to the computer trying to control deviations by adding warm water into the cooling jacket to increase the temperature if it falls below the set point and vice versa.

Note:
The numerous large spikes seen on the graph could be due to the high sensitivity of the probes or since this fermentation is done on a small scale with a low volume, any small changes may be reflected by big changes when plotted on a graph.
This graph also does not correspond to our growth curve that we plotted as this history plot belongs to another group’s.

Attention: There are additional questions to help in better understanding of this experiment. The question and answers are located in the next post.

Experiment 2 (Day 1-3): Further Questions

The answers below adresses the questions in our lab manual.

(1) On media preparation:

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

- Bacto-trptone is used to provide essential amino acids for the growing bacteria.
- Yeast extract is used to provide plethora of organic compounds required for bacteria growth.
- Sodium chloride provides sodium ions to E. coli for transport and osmotic balance.

b) What is the purpose of ampicillin?

It is to inhibit the growth of microbial contaminants.

c) Why is ampicillin added only after autoclaving?

If it is added before autoclaving, the ampicillin would be degraded when the autoclaving is done and render it ineffective.

(2) On equipment preparation:

a) What is meant by calibration of the pH probe?

The calibration process correlates the voltage produced by the probe. The calibration of the pH probe is performed with at least two standard calibration buffer solutions that are based on the range of pH values to be measured. Such calibration buffer solutions are available commercially of pH of 4.01, 7.00 and 10.00.

The probe must be calibrated at a zero point and a span point. Typically a probe will be calibrated at a pH of 7.0 and 10.0 or 4.01. The 7.0 calibration buffer solution is the Zero point calibration and the 10.0 or 4.01 calibration buffer is used for the Span adjustment. If you plan to measure pH of acidic solutions, calibration buffer pH 4.01 should be used and if you plan to measure pH of basic solutions, calibration buffer of 10.00 is used instead.

Calibration is done here by first placing the probe into the pH 7.00 buffer and allowing the pH reading to stabilize. For older pH meters, turn the calibration knob till pH 7.00 is shown, for modern pH meters just set it to calibration mode instead.

After calibrating at pH 7.00, rinse the probe with distilled water and do the same thing with the calibration buffers of pH 4.01 and/or pH 10.00. The pH probe should be rinsed with distilled water when moving from one buffer to the next.

Reference: http://www.ph-meter.info/pH-electrode-calibration

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

It is not suitable as a correction agent for pH because depending on temperature and agitation, hydrochloric acid at a concentration above 10% will produce a hydrogen chloride vapor that is very corrosive when combined with water vapour in the air. This corrosive vapour can corrode the metals in the fermenter. If hydrochloric acid is used, there must proper ventilation where the gases can be easily dissipated.

c) What is meant by polarization of the pO2 probe?

The pO2 probes contain 2 electrodes. They are the anode and the cathode which are both in contact with an electrolyte solution. The anode and the cathode are separated from the solution being measured, by an oxygen permeable membrane in which oxygen can diffuse through.

As oxygen passes by the membrane, reduction of oxygen occurs at the surface cathode which is exposed at the tip of the electrode. Oxygen molecules diffuse through the semi-permeable membrane and combine with the KCl electrolyte solution. The current produced is a result of the following reduction of oxygen at the cathode.

The pO2 probe measures oxygen tension amperometrically. The pO2 electrodes produce a current at a constant polarizing voltage which is directly proportional to the partial pressure of oxygen diffusing to the reactive surface of the electrode.

Reference: http://74.125.153.132/search?q=cache:bLvBdqF4Vv4J:www.enzyme.chem.msu.ru/eduproc/prac/taskA/Oxygen%2520Analyzers.doc+polarization+electrode+oxygen&cd=8&hl=en&ct=clnk&gl=sg

d) What is a peristaltic pump?

It is a type of positive displacement pump used for pumping a variety of fluid. It is used to remove liquid from the fermenter or to add acid or base, antifoaming reagent and nutrients to the culture gradually.

(3) On seed preparation:

a) What is the purpose of arabinose?

Arabinose is used as an inducer for the production of green fluorescent protein.

b) Describe the sterile techniques used in seed preparation.

The lab bench top was disinfected with 70% ethanol before the seed preparation was done. The bunsen burner was ignited to produce a flame for sterilizing of the cap of the cryovial (containing the bacteria culture) before opening it and to provide an aseptic zone to work in. A sterile plastic inoculating loop was used to inoculate the bacteria on to the agar plate. The agar plate cover was left half open when streaking was done instead of fully open to reduce the chance of contaminants from falling into the agar plate.

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

We perform step wise scale up to allow the cells to adapt to the culture conditions and grow to a large enough number to seed the fermenter. If we transfer the cell culture from the cryovial directly into the fermenter, there would be too little cells and the cells would not be able to grow or might have a very long lag phase.

Experiment 2 (Day 1-3)

Equipment, Media and Seed Culture Preparation

Objectives

1. To determine the steps to prepare a bioreactor for fermentation.
2. To prepare the media required for seed culture fermentation and scale-up fermentation.
3. To prepare seed culture required for scale-up fermentation.

Part I-Equipment Preparation Procedure

The steps below outline what was done by our TSO to prepare the various equipment required to carry out our fermentation.

Day 1

1) First we have to ensure the fermenter is clean and sterile. The whole fermenter was placed into a autoclave machine at 121 degree Celsius for 20minutes. However before doing so, we have to remove all the probes except the temperature probe as it would not be damaged during the autoclaving process. All silicon tubing should be clamped except those for exhaust filter and the female STT coupling of the sampling unit. Aluminum foil is then used to cover all filters and sockets to prevent condensation as these sites.

2) Certain probes require extra steps before they can be placed into the fermenter for use, such probes include the pH electrode probe, which require calibration using specific buffer solutions.

3) The pO2 probe on the other hand is required to be polarized for at least 6 hours, and later calibrated by aerating the probe with nitrogen.

4) When both probes and fermenter are ready, we can install the probes on the fermenter.
Certain probes such as the foam and level probe can be adjusted according to experimental needs.

The probes are also plugged into the machine at their specific jacks

Other accessories are added onto the fermenter, such as exhaust condensers, air inlet and exhaust filters and manual sampler unit.

The needs for such accessories are,

6) The additional reagent lines are then connected at this stage. Such addition reagents include, base (sodium hydroxide), acid (sulphuric acid) and anti-foam. The lines should pass through the peristaltic pumps, at the correction location. The use of such pumps aaaaaaallows fluid to be pumped slowly into the fermenter.

7) The picture below shows the peristaltic pumps. Selecting the Auto function allows the computer to control the amount being pumped which allows the computer to control the environment. Selecting the Manual function which is basically an “ON” switch, causes the respective reagent to be continuously pumped into the fermenter. Selecting the Off function which is basically an “OFF” switch deactivates the pump.

8) Now all the equipment is ready and the parameters that you wish to monitor can be set via the computer’s control panel.

a
Part II-Media Preparation Procedure

The steps below outline what we have done to prepare the media for the seed culture and the scale-up fermentation during our practical.

Day 1

1) 2 Litres of Luria-Bertani Medium were needed for the experiment (100ml for the shake flask and 1.9 Litres for the fermenter)
2) We used a pre-prepared Luria-Bertani Powder.

3) The content of the powder are as follows:

4) 50 grams of the powder were used to make the 2 liters of Luria-Bertani Medium.
5) 2 portions of 25g of powder was weighed and placed into a 2 liter bottle.

6) The bottle was topped up till the 2 liter mark with distilled water.

7) The mixture was then shaken till all the powder had dissolved.
8) Since powder took a while to dissolved, many people took turns to shake the mixture.
9) There was no need to adjust the pH of the Luria-Bertani Medium as the powder already adjusted the pH to 7.5. Also due to the fact that we were culturing bacteria cells (E. coli) which are more resistant to pH deviations from the optimum level.
10) The bottle was then labeled.

11) 100ml of the Luria-Bertani medium was transferred into a shake flask.
12) The shake flask was labeled.

13) Both shake flask and 2 litre bottle were autoclaved at 121 degree Celsius for 20 minutes and stored at 4 degree Celsius till needed.

a

Part III-Seed Culture Preparation Procedure

The steps below shows what we did during the 3 days to prepare a seed culture for the fermenter via scale-up fermentation.

Day 1

1) A cryovial of pGLO transformed E. coli was obtained from the -80oC freezer and thawed. Luria-Bertani Agar plates with ampicillin and arabinose were also obtained.

2) Using a disposable inoculating loop, the pGLO transformed E. coli were streaked onto the Luria-Bertani Medium with ampicillin 100ug/ml and arabinose 0.2% to obtain single colonies.
3) The bench was then swapped clean with 70% ethanol solution to sterilize it.

4) The Bunsen flame was ignited to ensure a sterile environment
5) The streaking can then begin.
6) The plate was labelled and left to incubate for 24 hours

Day 2

7) The agar plate which was plated the previous day was obtained. It was placed under UV light to view for any pGLO transformed E. coli growth.

However as you can see above, there was no cell growth; hence we were unable to use the plate to culture cells into the shake flask. There are many possible reasons which could account for the absence of cell growth. The E. coli cells provided to us may have not survived the freezing and thawing process or the culture in the vial was not mixed before streaking.


Luckily our dear TSO, Yong Hao had prepared a plate prepared for us in-case things like this happened.

In the image above you can see, his plate grew very well.

8) Now using disposable inoculation loop.
9) One of the group members took a big loop full of bacteria and inoculated it into the shake flask.

10) Note the Bunsen burner is also ignited, as to prevent contamination of the shake flask.
11) The shake flask was now left to incubate for 24 hours in a 32oC incubator.
12) The shake flask medium is made hence to be used for inoculation of the fermenter for a scale up fermentation.

Day 3

13) We obtained our shake flask from the incubator

As you can see from the image above, there was cell growth, as the medium was no longer clear when we first inoculated.

However based on his experience, Yong Hao suggested we used his flask which he probably had left to incubate for a longer time for inoculating the fermenter. This is because the cell growth in our shake flask was rather low; hence it might not be enough to do a proper inoculation on the fermenter.

14) Before inoculating the fermenter, Yong Hao assisted in adding ampicillin and arabinose into the Luria-Bertani Medium which he has transferred into the fermenter.

15) The ampicillin was added to prevent any unwanted organism or contaminants from growing in the fermenter, while arabinose was added to induce the production of GFP.
16) The culture from the shake flask was pumped into the fermenter for inoculation, which Ms Ang assisted us in.

Note the colour difference between the fermenter medium before and after inoculation

The fermenter was left to run on its own, while the computer and probes constantly measured any changes in the pH, Foam, temperature and dissolved oxygen level, and try to keep them constant.

Note: There are additional questions to help in better understanding of this experiment. The question and answers are located in the next post.

Experiment 1 (Day 1) : Further Questions

The questions found in our lab manual are adressed here.

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

Mammalian cells are more prone to damage by shear forces and hence require more gentle aeration and agitation as compared to microbial cells. Hence, the bioreactors used for mammalian cells are usually stirred tank with modified impellers (e.g. marine propeller type) at lower speeds of approximately 10-100 rpm and modified sparger for gentler aeration (e.g. bubbleless aeration).

Airlift fermenters or bubble columns maybe also used for mammalian cells to provide an environment even lower shear forces. Perfusion bioreactors used with immobilisation techniques are also used to provide a low shear force environment. (E.g. using micro-carriers or hollow fibres).

Since they are also more sensitive to deviations in culture conditions such as pH and dissolved oxygen, such parameters are more tightly regulated.

Microbial cells being more sturdy and are able to withstand higher shear forces and more extreme culture conditions are instead grown in standard stirred tank reactors with growth parameters not as tightly regulated.


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.

Experiment 1: familiarization with the bioreactor and its function.

We got to know about the location of different parts of the bioreactor, their names and function. We also took turns to learn how to do sampling.

Experiment 2: Equipment, media and seed culture preparation.

MEDIA PREPARATION:
We used powdered LB medium to make 2 litres of the media which to be used for culturing the transformed E. coli in the seed flask and in the fermenter. After autoclaving, ampicillin and arabinose were added to the medium in the fermenter.

SEED CULTURE PREPARATION:
Streaking of pGLO transformed Escherichia coli from a thawed cryovial on LB/Amp/Ara plate was first done. After incubation, several colonies of pGLO transformed E.coli from a fresh LB/Amp/Ara plate was transferred to a shake flask containing LB medium with ampicillin and incubated.

EQUIPMENT PREPARATION:
The Fermenter was sterilised and the various probes were calibrated and installed in the fermenter and connected to the computer. Additional accessories (exhaust condensers, air filters and manual sampler unit) are also installed. Reagent bottles (containing antifoam, acid and base) are hooked up to the fermenter via the reagent lines which will run through the peristaltic pumps. Finally, parameters are set via the control panel and the fermenter is ready for use.

Note: Equipment preparation was done by our TSO, Mr Wee Yong Hao. :)

Experiment 3: Inoculation, fermentation and monitoring

100ml of the seed culture from the shake flask was inoculated into the culture medium in the fermenter using a pump. Control parameters are set, and the fermentation was allowed to take place for 24hours. The readings of the control parameters were plotted on a graph by the computer over the course of the fermentation. This graph was analysed later.

For the first 10 hours of fermentation, samples of the culture broth in the fermenter were taken every hour. The absorbance of these samples were taken later and analysed.

Experiment 4: isolation and purification of product

ISOLATION:
Green fluorescent product is an intracellular product; hence the bacteria cells need to be lysed first to release the protein. 3 methods of cell disruption (using enzymes, freezing and thawing, and sonication) were performed on the bacteria cells. After cell disruption, the extract is obtained from the supernatant after centrifuging.

PURIFICATION:
The extract that was obtained from isolation was purified using size exclusion chromatography. After purification, the absorbance reading of 8 fractions and blank was taken using the spectrophotometer set at 476 nm, recorded and analysed.

Experiment 1 (Day 1)

Familiarization with the Bioreactor and its Operation

Objectives:

• To identify the various parts and the components of the microbial and mammalian fermenters.
• To understand the purpose of the different parts of the bioreactor.
• To understand the basic operation procedures of a bioreactor.

During the very first practical, were familiarised with the bioreactor and introduced its different parts and functions by Ms Ang. The table and labelled diagram below summarises what we have learnt in this practical.

Picture Obtained from Lab Manual

Left-Control Panel; Right-Inlet Jacks for Various Probes

Take a look at the slide show below showing the labeled parts of our fermenter.

Note: There are additional questions to help in better understanding of this experiment. The question and answers are located in the next post.

How GFP is Incorporated in E. coli

In order to allow E. coli to produce GFP protein, genetic engineering has to be carried out to insert the GFP gene into E. coli cells. In order for this to be done the GFP gene has to be first introduced into vectors which will then be incorporated into E.coli.

In this case, the vector used are small circular DNA molecules that replicate independently of the genome called plasmids. The plasmids used have been modified to function as vectors, often containing a seletable biomarker gene as such that of antibiotic resistance (eg. Ampicillin resistance) apart from the gene of interest to be introduced. The biomarker gene serves an important role of allowing successfully transformed bacteria to be selected from the unsuccessful ones.

The plasmid used to create transformed E.coli which were used in our fermentation experiment is Biorad’s pGLO plasmid which contains a biomarker gene for ampicillin resistance and a gene coding for the green fluorescent protein. The pGLO also incorporates a gene regulatory system, which activates transcription of GFP in the pressence of the sugar, arabinose.

The pGLO can be introduced into E. coli by a variety of transformation methods. One of these methods is by using calcium chloride. E. coli cells and pGLO are treated with a weak solution of calcium chloride under low temperatures. The positively charged calcium ions are thought to bind to the negatively charged cell wall and allow the pGLO plasmid to adsorb onto the bacteria surface. The E. coli and pGLO are then heat shocked to allow the pGLO to enter E. coli causing it to become transformed.

Since the pGLO plasmid contains both the regulatory sequence together with the GFP gene and the gene for ampicillin resistance, sucessful transformants can be selected by growing these E. coli cells on LB agar containing ampicillin and arabinose. Sucessful transformants will be able to grow on the agar plate and transformants containing the GFP gene will be able to express GFP due to the pressence of Arabinose.

References

• http://iiumedic.com/web/v1/wp-content/uploads/2009/08/5-bactransformation.pdf
• http://www.clt.astate.edu/agrippo/fall2005lab7pGLO%20Transformation.doc
• http://whyfiles.org/246e_coli/images/e_coli.jpg
• http://orgchem.colorado.edu/hndbksupport/drying/images/decant.jpg
• http://cba.mit.edu/docs/04.05.NSF/images/green.jpg
• http://www.cvgs.k12.va.us/research/Final/sresch06/arrington/pglomap_resized.jpg
• http://unlmbsc.files.wordpress.com/2007/11/pglo-for-blog.jpg

Theory Behind Green Flurescent Protein (GFP)

The green fluorescent protein or GFP which was first isolated from Aequorea Victoria (jellyfish species), is able to give off green colour fluorescent light when illuminated with ultraviolet light (UV).

Green Fluorescence is produced naturally in Aequorea Victoria when calcium binds to a protein, aequorin causing it to release blue light. This blue light is then absorbed by GFP and emitted as green light. GFP from Aequorea Victoria has an excitation peaks at wavelengths of 395nm and 475nm where the GFP absorbs light causing its electrons to become excited and emit green light at 509nm.

(Left-Topology of GFP; Right-GFP Structure with Chromophore in the center)

GFP is 26.9kDa in size and has a unique beta-can structure made of 11 beta-strands which forms a beta-barrel with an alpha-helix running through the center. The chromophore (a p-hydroxybenzylideneimidazolinone) which is responsible for the fluorescent property of GFP is formed from residues 65–67, which are Ser-Tyr-Gly in native GFP.

GFP can be used in applications such as biomarkers or bioreporters where the GFP gene could be incorporated into genes encoding for proteins and be translated out together with the protein of interest. This allows the location and the movement of the protein to be detected. This can serve to be a useful tool as it allows us to determine what are the protein’s target site in the body or when they are made. Another advantage that GFP possess is that unlike other small fluorescent molecules used previously, GFPs are less harmful to living cells when illuminated. Hence GFP is a valuable marker of gene expression.

References

• http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
• http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/pps99.cryst.bbk.ac.uk/projects/gmocz/gfp.html
• http://lem.ch.unito.it/didattica/infochimica/2008_GFP/struttura.html
• http://www.rpc.msoe.edu/cbm2/gfp1.htmhttp://www.nature.com/nature/journal/v440/n7082/full/440280a.html
• http://klemkelab.ucsd.edu/images/research/proteomics/fig4.gif
• http://www.uib.no/imagearchive/stort-hovedtekstbilde_Slide4.jpg