BIOPROCESS TECHNOLOGY GROUP REPORT
- Monitoring the entire fermentation process. - Purify the Green Fluorescence Protein (GFP). - Obtain the final product of glowing e. coli in UV light.
Introduction
Have a look at the jellyfish, doesn't it look beautiful?! So what exactly causes the jellyfish to radiate a green glow??
Well, the answer is Green Fluorescent Protein (GFP). GFP is a naturally fluorescent protein originally isolated from a jellyfish, named Aequorea victoria.
It is a 238 amino acid protein that is responsible for the bioluminescence of this organism. The GFP gene can be introduced into cultured cells or into specific cells. These cells will then be able to express the gene, emitting green fluorescence, when exposed to UV light.
This makes GFP a useful marker for studying gene expression and the location of proteins. Since proteins are extremely small, linking GFP to a specific protein can allow one to see when a protein is synthesized, in the cell, under ultra-violet light.
Now, how cool is that?!!
The GFP gene can be incorporated into the bacterial plasmid, to produce DNA fragments containing the GFP gene after replication.
Isolated plasmid DNA from Escherichia coli is used as the cloning vector. In addition, the genomic DNA of the jellyfish is isolated too. After the isolation of the genomic DNA and plasmid vector, it must be cut into pieces before the chromosomal DNA and the plasmid vector can be joined. This process is called restriction digestion.
Using restriction enzyme, it only cuts at specific sites. Thus, the restriction enzyme cuts the genomic DNA of the jellyfish at the GFP sites and cuts the plasmid DNA at sites where the GFP gene will be incorporated.
After successfully cleaving the genomic DNA and the plasmid vector, the cut ends are known as sticky ends. These ends have the ability to form bond with another piece of DNA. Following restriction digestion is ligation. This involves joining linear DNA fragments together with covalent bonds. The ends of both GFP gene and the plasmid vector are complementary and sticky. Creating a bond at these sites results in one continuous, circular piece of DNA.
Once a recombinant plasmid with the gene of interest is formed, it is then introduced into a host, which is a bacteria cell. This process is known as transformation. When the bacterial cells replicate, the plasmid containing the gene of interest also replicates. The bacteria will grow, divide and produce many identical copies of the recombinant DNA.
_ _ _ _ _ _LiFe being mushr00miSh ( ^.^ )
Copyright© 2007 Nanyang Polytechnic Diploma in Molecular Biotechnology MB0603 Bioprocess Technology Group 3A. All rights reserved.
Objectives - To familiarize students with the parts and components of microbial and mammalian bioreactors. - To introduce the basic operation procedure of a bioreactor.
A very important and common question asked when it comes to understanding a bioreactor, especially for people who have never heard such a term is, “what in the world is a bioreactor?” A bioreactor is a vessel in a chemical process which involves organisms or biochemically active substances derived from such organisms is carried out. The next question inline to be asked is, “How do these machines or reactors look like?” Bioreactors are commonly cylindrical, ranging in size from some liter to cube meters, and are often made of stainless steel.
Cell culture bioreactors are categorized into two types:- Those that are used for cultivation of anchorage dependent cells (e.g. primary cultures derived from normal tissues and diploid cell lines.) - Those that are used for the cultivation of suspended mammalian cells (e.g. cell lines derived from cancerous tissues and tumours, transformed diploid cell lines, hybridoma).
However unlike mammalian cells, for suspended non-mammalian cells, they are cultured in different ways. For example, plant cell culture methods, bacterial culture methods, and viral culture methods. They need either a solid medium, usually a gel such as agar, or a liquid medium to grow. In some cases the bioreactor may be modified to grow both anchorage dependent and suspended cells. Ideally any cell culture bioreactor must maintain a sterile culture of cells in medium conditions which maximize cell growth and productivity. The type of bioreactor used in this experiment is for suspended cell culture. Basically, in this first part of the experiment, we were told to get familiarized with the bioreactor. As a group, we studied the various parts of the fermenter like the motor, impeller, sparger, baffles and many more. Then we were asked to explain the functions of each part with the help of our lecturer. We also did a streak plate of the E.coli to be used.
Diagram of Fermentor Diagram 1 - Pictorial Representation of a Fermentor
Diagram 2 - Fermentor used in the experiment
Diagram 3 - Fermentor connected to the Control Panel
Functions of the indivual parts
Discussion
For this experiment, we needed several colonies of pGLO transformed E.coli to be transferred to the flask containing 100ml LB medium with ampicillin.
Therefore, streaking was done on a LB/Amp/Ara plate using pGLO transformed E.coli so that the inoculum will be progressively ‘thinned out’. This means that isolated, individual colonies of pGLO transformed E.coli can be obtained.
Questions 1. State the differences you observe between a microbial bioreactor and a mammalian cell bioreactor.
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.
First stage - Get familiarized with the different parts of the fermentor and their functions.
Second stage - The Luria-Bertani Medium was prepared. - The pH electrode was calibrated using standard buffer solution. - The pH probe, pO2probe, foam, level probe, exhaust condensers, air inlet and exhaust filters were installed. - E. coli was streaked on a LB/Amp/Ara plate by the streak plate method.
Third stage - Ampicillin and arabinose were added to the culture medium. - The control parameters were adjusted. - The fermenter was inoculated with 100 ml of seed culture and 10 ml of sample was taken out every hour.
Fourth stage - Isolation of Green Fluorescent Protein was done by using enzymes, freezing and thawing and sonication. - Purification of product was done using gel permeation chromatography. - Absorbance reading was then taken using the spectrophotometer.
_ _ _ _ _LiFe of a Shr00m >.<
Objectives – 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
Alright, we all know what fermentation is. It is basically to help in the conversion of a substrate into some useful products by the help of microorganisms. A simple example would be the making of beer using yeast as the microorganism. Even though yeast stinks, the final product is pretty good! We need to prepare 3 very important things. It would include the media, the equipment which is the bioreactor of course and lastly the cells which are also known as the seed culture. These 3 very important things should be closely examined before the process begins. The media should contain all the appropriate ingredients needed, the bioreactor has to be sterilized or else we’re gona get products that are contaminated and lastly the cells should be grown to an optimum growth condition.
Procedures Equipment 1. The pH electrode was calibrated using the buffer solution 2. pH probe, pO2 probe, foam and level probe were installed into the top plate 3. The agent lines for acid, base and antifoam were connected and other accessories were also installed 4. Then the bioreactor was prepared for sterilization and was autoclaved for 20 minutes 5. Lastly, pO2 electrode was polarized for about 6 hours Media 1. Firstly, 2 litres of LB media was prepared 2. 500ml of the media was transferred into a shaker flask, while the remaining was transferred to the 2litre bioreactor 3. Then it was autoclaved for 20 minutes 4. Ampicillin was added to the media after cooling process and the media was kept at 4ºC till inoculation Culture 1. pGLO transformed E.coli was retrieved from the freezer 2. The E.coli was then streaked on a LB/Amp/Ara plate and left to incubate for a day. 3. Several colonies of the transformed E.coli were transfered to a flask containing 100ml LB medium with ampicillin. 4. The flask was placed in the shaking incubator and was left to incubate for 24 hours at 32ºC. 5. Lastly, it was used to inoculate the fermenter for scale-up fermentation.
That's us preparing the stinky broth.
The making of the broth.
Watch us glow!!
In Day 2’s practical, we had to put the colonies from the agar plates into the 100ml of broth in the shake flask culture. This is to allow for the bacteria to grow to a certain number, so that the lag phase of the culture will not be too long, as a long lag phase would mean that the culture would produce the GFP at a longer time. However, the cells in our flask grew too slowly, and after 24 hours, there was still hardly any growth in the shake flask. Therefore, when we seed the shake flask culture into the fermentor, we added more cells from the agar and from the vial. Thus, this will prolong the time for lag phase in the fermentor when the cells are adapting to the conditions in the fermentor.
Questions 1. On media preparation:
a. Explain the purpose of each ingredient found in the LB media. – Bacto-tryptone: to provide the essential amino acids (peptones and peptides), carbon, energy and many macro and micronutrients for bacterial growth. – Yeast extract: to provide a surplus of organic compounds, such as vitamins and certain trace elements, useful for bacterial growth. – Sodium Chloride: 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 ampicillin? – It is a beta-lactam antibiotic that inhibits the growth of other bacteria than the desired bacteria, hence preventing contamination.
c. Why is ampicillin added only after autoclaving? – It will be inactivated when exposed to high heat, therefore it shouldn’t be added into a very warm solution and the solution it is in shouldn’t be reheated.
2. On equipment preparation:
a. What is meant by calibration of the pH probe? – It is to perform an optimal pH of media with the use of at least two buffers, one is at pH 7.0 (neutral) and the other is pH 4.0 (acidic) and pH 10.0 (basic).
b. Why is hydrochloric acid not suitable as a correction agent for pH? – Hydrochloric acid is not suitable as a correction agent for pH as it is a very strong acid and thus can fully dissociate in water and affecting the optimum composition of the fermenter by removing water from the solution.
c. What is a peristaltic pump? – A peristaltic pump is a type of positive displacement pump used for pumping a variety of fluids. The fluid is surrounded by a flexible tube fitted inside a circular pump casing. A rotor with a few 'rollers' are attached to the external circumference compresses the flexible tube. In this experiment, the addition of antifoam agents is pumped in via peristaltic pumps.
3. On seed preparation:
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. It also “turn” on the cells that have the pGLO genes that code for the fluorescent protein and this results in a fluorescent colour being produced when the cells which have the pGLO genes grown in the arabinose.
b. Describe the sterile techniques used in seed preparation. – Sterile techniques was used in seed preparation by wearing gloves when several colonies of pGLO transformed E. coli was transferred from a fresh LB/Amp/Ara plate to the flask containing 100 ml LB medium with ampicillin in the fume hood. This is to prevent any contaminants from the hands to cause contaminations. pGLO transformed E. coli was also retrieved from the -80oC freezer as most of the microbes would not be able to survive under such low temperature and therefore the chances of contamination is greatly reduced.
c. Why do we perform step-wise scale-up instead of transferring directly to the fermenter? – A step-wise scale-up is performed instead of transferring directly to the fermenter so as to ensure that all the contaminants in the fermenter are being removed and not left behind to interfere with the results and thus prevent contamination.
Objectives – To carry out scale-up fermentation process to increase the yield of Green florescent protein. – To monitor cell growth and product formation through manual sampling and computer data logging.
Fermentor: The general idea behind a fermentor is to provide a stable and optimal environment for bacteria, in which they can reproduce and it also includes those used to grow large quantities of genetically engineered bacteria. These bacteria, having had the genes that code for various proteins spliced into them, will grow and reproduce, and will express the inserted gene, as for this experiment. This will results in the desired protein being released into the growth medium, from where it can be harvested, purified, and then used.
Microorganisms: Industrial microorganisms are organisms that produce a desired product, thus they are initially selected from natural samples. However, the strain is then modified to improve the product yield. The selected strain is unlikely to survive well in nature. Other desirable characteristics of industrial microbes are (1) rapid growth, (2) genetic stability, (3) non-toxicity to humans, and (4) large cell size, for easy removal from the culture fluid.
Products: The aim of Industrial microbiologists is to produce (1) microbial biomass (2) specific enzymes, or (3) metabolites. Metabolites may be major metabolic products of catabolism. Industrial microbiologists culture organisms in many of the same ways as other microbiologists, but the goal is often to produce very large quantities. Below is the equation of what microbes require for fermentation and what they give out as products.
Carbon & energy source + nitrogen source + O2 + other requirements → Biomass + Product + byproducts + CO2 + H2O + heat
Fermentation conditions: The capacity of the bioreactor used in this experiment is 2L.. The process is aerobic. The fermentor has an external jacket by which it can be sterilized initially and cooled during the fermentation. Spargers and impellers in the vessel are used for aeration and stirring of the contents. The vessel may contain various devices for monitoring the environmental conditions within the culture, so that these factors can be controlled to obtain high product yields.
Inoculum: The organisms used in industrial processes must be carefully preserved in liquid nitrogen. The inoculum for the fermentor must be built up from a working strain. Since inoculum should be 5-10% of the culture volume, the inoculum for the fermentor is 100ml.
Procedures Setting Up - The ampicillin and arabinose stock solutions were already prepared and filter-sterilized. - The medium broth was cooled to below 50° C and ampicillin was added to a final concentration of 100ug/ml and arabinose to a final concentration of 0.2%. The control parameters were set as shown:
The fermentor was inoculated with 100ml of seed culture. Followed by that, the fermentation was continued for 24 hours at the above conditions.
TSO is fixing up our fermentor! Monitoring of the Fermentation Process 10ml of blank sample was taken before the inoculation and a sample, consisting of 10ml, was taken for every hour. The fermentation broth was harvested after 24 hours of fermentation. 10ml of culture was then aliquot into a sterile, disposable test tube. Constance & YihLin are taking the 1st sample!!! Results o Culture absorbance, fermentation properties o Graph of Cell Growth Lag Phase: Bacteria are trying to adapt to the new environmental conditions to which they have been introduced such as pH, temperature and nutrients. This is the period where individual bacteria are maturing and not yet able to divide. There is no significant increase in numbers with time. Exponential Growth Phase: The living bacteria population increases rapidly with time at an exponential growth in numbers, and the growth rate increasing with time. Conditions are optimal for growth. Stationary Phase: With the exhaustion of nutrients as well as the increase in waste products, the growth rate has slowed to the point where the growth rate equals to the death rate. Effectively, there is no net growth in the bacteria population. Death Phase: Due to the lack of nutrient, the living bacteria population decreases with time. o History plot of the major operational procedures Discussion Graph Discussion will be based on the log (X/Xo) against time graph since both graphs are similar. As you can see, for the first 5 hours or so, the graph is basically crawling on the X-axis. This is actually the lag phase (an extremely long one). Generally, the lag phase for E.coli should be around 1 to 2 hours. As explained earlier, this long lag phase results from adding the cells from the agar plate directly to the fermentor vessel. During lag phase, the cells are adapting to their new environment. Enzymes are made to utilize the new nutrients found in the new environment. Therefore, growth will not be expected in this phase. The length of the lag phase depends on the conditions the E.coli has previously existed in and the conditions the E.coli is living in at present. Apart from the reason given earlier, a long lag phase may also be resulted when previously: – The cells were in harsh conditions – They were grown with different nutrients and different temperature From T = 5h onwards, the graph rises almost steadily. This is the logarithmic phase (although the number of cells did not really increase exponentially). This phase occurs because the cells have adapted to the environment already. Hence, they started to grow and divide at the maximum rate which they can have under the conditions given. Since GFP protein is a primary metabolite; it should be actively produced during the logarithmic phase. Due to the long lag phase, the stationary phase and death phase of the growth curve could not be observed in the limited hours of the experiment. Although the last two phases cannot be observed, explanations on the phases shall be given for those who know nothing about these. Stationary phase happens when cell growth = cell death. This is because the nutrients are used up as the cells grow (no addition of nutrients since this is a batch culture) and waste produced by the cells are accumulated (no removal of waste since this is a batch culture). Due to the lack of nutrients and accumulated waste, cell growth slows down. Death phase occurs when the cell growth < cell death. As the stationary phase continues, nutrients will eventually be depleted and the medium will be filled with toxic waste products. The conditions of the medium become very unfavorable for the cells. Therefore, cells start to die faster than they grow. In this experiment, the number of cells was not determined. This is because the main objective of the experiment was to let the cells grow so that the GFP protein can be produced and harvested. Also, the absorbance value is directly proportional to the number of cells (number of cells increase, absorbance value increases). Therefore, the absorbance values can be used to determine whether the cells are growing. However, the number of cells can be determined (if you want). A viable cell count can be made at, maybe, T = 5. At the same time, take the absorbance reading. By doing so, we will roughly know the number of cells for a certain absorbance value. Hence, after taking all the absorbance values, calculations can be made so that the estimated number of cells for each value can be known. History Plot From the plot, we can see the temperature values (blue), stirring rate (green), PO2 values (teal), and pH values (red). It can be seen that, basically, the lines fluctuates a lot. As cells grow, they use up the nutrients, oxygen etc. Heat will be produced due to metabolism. pH will be decreased due to carbon dioxide given off. The system needs to adjust the parameters constantly to ensure that the cells are growing in their optimum conditions. Therefore, fluctuations of the values of the different parameters were seen. However, the general trends of the parameters were stable. This meant that the system is working well and hard making the conditions optimal for cell growth. Questions 1. Explain the control philosophy for pH, temperature and dissolved oxygen as was used in the fermentation process. pH– pH is measured by the pH probe. Readings of pH are taken constantly, and if the system detects a change in pH, away from the optimum of pH 7.5, it will automatically pump in either acid or base if the pH is increasing or if the pH is decreasing, to increase or decrease the pH respectively, till the pH returns to the optimum level. Acid used in the acid pump is sulphuric acid, H2SO4, and base used in the base pump is sodium hydroxide, NaOH Temperature– Temperature is measured by the temperature probe. If the temperature increases from the optimum of 32°C, water will be pumped through the cooling jet to lower the temperature. Dissolved Oxygen– Dissolved oxygen in the fermentor culture solution is measured by the dissolved oxygen probe. If the amount of dissolved oxygen is lower than 25%, air will be allowed into the fermentor, via the sparger. 2. Describe the principle of the spectrophotometer which was used to determine the cell density (OD600). Why was 600nm used? – Spectrophotometer is employed to measure the amount of light a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The filtered light is then absorbed by the absorbing substance (analyte) in the sample. Therefore, if the absorbance value is low, it means that lesser light is being absorbed by the analyte and thus indicates that the cell density is lower. In contrast, if the absorbance value is high, it means that more light is being absorbed by the analyte and thus the cell density will be higher. − 600 nm was used instead as Green Fluorescent Protein has an emission peak of more than 500 nm, which is around 509 nm as shown in the lower green portion of the visible portion below. 3. Is GFP a primary or secondary metabolite? At which phase should the product be harvested? At which phase was the product actually harvested? – Green Fluorescent Protein is a primary metabolite that is produced during primary growth phase, during lag, and log phase. It should be harvested during stationary phase. However, it was collected during death phase. 4. What are some advantages of using a computer control system? From the history char (which will be given to you by your supervisor after the fermentation), comment on the effectiveness of the computer control. – Some of the advantages of using a computer control system are that it can eliminate the need for creating an archive of nutrient consumption rate, pH values, dissolved oxygen and temperature profiles. It also allows automatic control over the maintenance of nutrient level within a narrow range, pH values, dissolved oxygen and temperature values. _ _ _ _ _LiFe of a Shr00m >.<
Monitoring of the Fermentation Process 10ml of blank sample was taken before the inoculation and a sample, consisting of 10ml, was taken for every hour. The fermentation broth was harvested after 24 hours of fermentation. 10ml of culture was then aliquot into a sterile, disposable test tube.
Constance & YihLin are taking the 1st sample!!! Results o Culture absorbance, fermentation properties o Graph of Cell Growth Lag Phase: Bacteria are trying to adapt to the new environmental conditions to which they have been introduced such as pH, temperature and nutrients. This is the period where individual bacteria are maturing and not yet able to divide. There is no significant increase in numbers with time. Exponential Growth Phase: The living bacteria population increases rapidly with time at an exponential growth in numbers, and the growth rate increasing with time. Conditions are optimal for growth. Stationary Phase: With the exhaustion of nutrients as well as the increase in waste products, the growth rate has slowed to the point where the growth rate equals to the death rate. Effectively, there is no net growth in the bacteria population. Death Phase: Due to the lack of nutrient, the living bacteria population decreases with time. o History plot of the major operational procedures Discussion Graph Discussion will be based on the log (X/Xo) against time graph since both graphs are similar. As you can see, for the first 5 hours or so, the graph is basically crawling on the X-axis. This is actually the lag phase (an extremely long one). Generally, the lag phase for E.coli should be around 1 to 2 hours. As explained earlier, this long lag phase results from adding the cells from the agar plate directly to the fermentor vessel. During lag phase, the cells are adapting to their new environment. Enzymes are made to utilize the new nutrients found in the new environment. Therefore, growth will not be expected in this phase. The length of the lag phase depends on the conditions the E.coli has previously existed in and the conditions the E.coli is living in at present. Apart from the reason given earlier, a long lag phase may also be resulted when previously: – The cells were in harsh conditions – They were grown with different nutrients and different temperature From T = 5h onwards, the graph rises almost steadily. This is the logarithmic phase (although the number of cells did not really increase exponentially). This phase occurs because the cells have adapted to the environment already. Hence, they started to grow and divide at the maximum rate which they can have under the conditions given. Since GFP protein is a primary metabolite; it should be actively produced during the logarithmic phase. Due to the long lag phase, the stationary phase and death phase of the growth curve could not be observed in the limited hours of the experiment. Although the last two phases cannot be observed, explanations on the phases shall be given for those who know nothing about these. Stationary phase happens when cell growth = cell death. This is because the nutrients are used up as the cells grow (no addition of nutrients since this is a batch culture) and waste produced by the cells are accumulated (no removal of waste since this is a batch culture). Due to the lack of nutrients and accumulated waste, cell growth slows down. Death phase occurs when the cell growth < cell death. As the stationary phase continues, nutrients will eventually be depleted and the medium will be filled with toxic waste products. The conditions of the medium become very unfavorable for the cells. Therefore, cells start to die faster than they grow. In this experiment, the number of cells was not determined. This is because the main objective of the experiment was to let the cells grow so that the GFP protein can be produced and harvested. Also, the absorbance value is directly proportional to the number of cells (number of cells increase, absorbance value increases). Therefore, the absorbance values can be used to determine whether the cells are growing. However, the number of cells can be determined (if you want). A viable cell count can be made at, maybe, T = 5. At the same time, take the absorbance reading. By doing so, we will roughly know the number of cells for a certain absorbance value. Hence, after taking all the absorbance values, calculations can be made so that the estimated number of cells for each value can be known. History Plot From the plot, we can see the temperature values (blue), stirring rate (green), PO2 values (teal), and pH values (red). It can be seen that, basically, the lines fluctuates a lot. As cells grow, they use up the nutrients, oxygen etc. Heat will be produced due to metabolism. pH will be decreased due to carbon dioxide given off. The system needs to adjust the parameters constantly to ensure that the cells are growing in their optimum conditions. Therefore, fluctuations of the values of the different parameters were seen. However, the general trends of the parameters were stable. This meant that the system is working well and hard making the conditions optimal for cell growth. Questions 1. Explain the control philosophy for pH, temperature and dissolved oxygen as was used in the fermentation process. pH– pH is measured by the pH probe. Readings of pH are taken constantly, and if the system detects a change in pH, away from the optimum of pH 7.5, it will automatically pump in either acid or base if the pH is increasing or if the pH is decreasing, to increase or decrease the pH respectively, till the pH returns to the optimum level. Acid used in the acid pump is sulphuric acid, H2SO4, and base used in the base pump is sodium hydroxide, NaOH Temperature– Temperature is measured by the temperature probe. If the temperature increases from the optimum of 32°C, water will be pumped through the cooling jet to lower the temperature. Dissolved Oxygen– Dissolved oxygen in the fermentor culture solution is measured by the dissolved oxygen probe. If the amount of dissolved oxygen is lower than 25%, air will be allowed into the fermentor, via the sparger. 2. Describe the principle of the spectrophotometer which was used to determine the cell density (OD600). Why was 600nm used? – Spectrophotometer is employed to measure the amount of light a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The filtered light is then absorbed by the absorbing substance (analyte) in the sample. Therefore, if the absorbance value is low, it means that lesser light is being absorbed by the analyte and thus indicates that the cell density is lower. In contrast, if the absorbance value is high, it means that more light is being absorbed by the analyte and thus the cell density will be higher. − 600 nm was used instead as Green Fluorescent Protein has an emission peak of more than 500 nm, which is around 509 nm as shown in the lower green portion of the visible portion below. 3. Is GFP a primary or secondary metabolite? At which phase should the product be harvested? At which phase was the product actually harvested? – Green Fluorescent Protein is a primary metabolite that is produced during primary growth phase, during lag, and log phase. It should be harvested during stationary phase. However, it was collected during death phase. 4. What are some advantages of using a computer control system? From the history char (which will be given to you by your supervisor after the fermentation), comment on the effectiveness of the computer control. – Some of the advantages of using a computer control system are that it can eliminate the need for creating an archive of nutrient consumption rate, pH values, dissolved oxygen and temperature profiles. It also allows automatic control over the maintenance of nutrient level within a narrow range, pH values, dissolved oxygen and temperature values. _ _ _ _ _LiFe of a Shr00m >.<
Results o Culture absorbance, fermentation properties
o Graph of Cell Growth
Lag Phase: Bacteria are trying to adapt to the new environmental conditions to which they have been introduced such as pH, temperature and nutrients. This is the period where individual bacteria are maturing and not yet able to divide. There is no significant increase in numbers with time. Exponential Growth Phase: The living bacteria population increases rapidly with time at an exponential growth in numbers, and the growth rate increasing with time. Conditions are optimal for growth. Stationary Phase: With the exhaustion of nutrients as well as the increase in waste products, the growth rate has slowed to the point where the growth rate equals to the death rate. Effectively, there is no net growth in the bacteria population. Death Phase: Due to the lack of nutrient, the living bacteria population decreases with time.
o History plot of the major operational procedures
Graph
Discussion will be based on the log (X/Xo) against time graph since both graphs are similar.
As you can see, for the first 5 hours or so, the graph is basically crawling on the X-axis. This is actually the lag phase (an extremely long one). Generally, the lag phase for E.coli should be around 1 to 2 hours. As explained earlier, this long lag phase results from adding the cells from the agar plate directly to the fermentor vessel.
During lag phase, the cells are adapting to their new environment. Enzymes are made to utilize the new nutrients found in the new environment. Therefore, growth will not be expected in this phase. The length of the lag phase depends on the conditions the E.coli has previously existed in and the conditions the E.coli is living in at present. Apart from the reason given earlier, a long lag phase may also be resulted when previously: – The cells were in harsh conditions – They were grown with different nutrients and different temperature
From T = 5h onwards, the graph rises almost steadily. This is the logarithmic phase (although the number of cells did not really increase exponentially). This phase occurs because the cells have adapted to the environment already. Hence, they started to grow and divide at the maximum rate which they can have under the conditions given. Since GFP protein is a primary metabolite; it should be actively produced during the logarithmic phase.
Due to the long lag phase, the stationary phase and death phase of the growth curve could not be observed in the limited hours of the experiment.
Although the last two phases cannot be observed, explanations on the phases shall be given for those who know nothing about these.
Stationary phase happens when cell growth = cell death. This is because the nutrients are used up as the cells grow (no addition of nutrients since this is a batch culture) and waste produced by the cells are accumulated (no removal of waste since this is a batch culture). Due to the lack of nutrients and accumulated waste, cell growth slows down.
Death phase occurs when the cell growth < cell death. As the stationary phase continues, nutrients will eventually be depleted and the medium will be filled with toxic waste products. The conditions of the medium become very unfavorable for the cells. Therefore, cells start to die faster than they grow.
In this experiment, the number of cells was not determined. This is because the main objective of the experiment was to let the cells grow so that the GFP protein can be produced and harvested. Also, the absorbance value is directly proportional to the number of cells (number of cells increase, absorbance value increases). Therefore, the absorbance values can be used to determine whether the cells are growing.
However, the number of cells can be determined (if you want). A viable cell count can be made at, maybe, T = 5. At the same time, take the absorbance reading. By doing so, we will roughly know the number of cells for a certain absorbance value. Hence, after taking all the absorbance values, calculations can be made so that the estimated number of cells for each value can be known.
History Plot From the plot, we can see the temperature values (blue), stirring rate (green), PO2 values (teal), and pH values (red).
It can be seen that, basically, the lines fluctuates a lot. As cells grow, they use up the nutrients, oxygen etc. Heat will be produced due to metabolism. pH will be decreased due to carbon dioxide given off. The system needs to adjust the parameters constantly to ensure that the cells are growing in their optimum conditions. Therefore, fluctuations of the values of the different parameters were seen.
However, the general trends of the parameters were stable. This meant that the system is working well and hard making the conditions optimal for cell growth.
Questions 1. Explain the control philosophy for pH, temperature and dissolved oxygen as was used in the fermentation process.
pH– pH is measured by the pH probe. Readings of pH are taken constantly, and if the system detects a change in pH, away from the optimum of pH 7.5, it will automatically pump in either acid or base if the pH is increasing or if the pH is decreasing, to increase or decrease the pH respectively, till the pH returns to the optimum level. Acid used in the acid pump is sulphuric acid, H2SO4, and base used in the base pump is sodium hydroxide, NaOH Temperature– Temperature is measured by the temperature probe. If the temperature increases from the optimum of 32°C, water will be pumped through the cooling jet to lower the temperature. Dissolved Oxygen– Dissolved oxygen in the fermentor culture solution is measured by the dissolved oxygen probe. If the amount of dissolved oxygen is lower than 25%, air will be allowed into the fermentor, via the sparger.
2. Describe the principle of the spectrophotometer which was used to determine the cell density (OD600). Why was 600nm used? – Spectrophotometer is employed to measure the amount of light a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The filtered light is then absorbed by the absorbing substance (analyte) in the sample. Therefore, if the absorbance value is low, it means that lesser light is being absorbed by the analyte and thus indicates that the cell density is lower. In contrast, if the absorbance value is high, it means that more light is being absorbed by the analyte and thus the cell density will be higher. − 600 nm was used instead as Green Fluorescent Protein has an emission peak of more than 500 nm, which is around 509 nm as shown in the lower green portion of the visible portion below.
3. Is GFP a primary or secondary metabolite? At which phase should the product be harvested? At which phase was the product actually harvested? – Green Fluorescent Protein is a primary metabolite that is produced during primary growth phase, during lag, and log phase. It should be harvested during stationary phase. However, it was collected during death phase.
4. What are some advantages of using a computer control system? From the history char (which will be given to you by your supervisor after the fermentation), comment on the effectiveness of the computer control. – Some of the advantages of using a computer control system are that it can eliminate the need for creating an archive of nutrient consumption rate, pH values, dissolved oxygen and temperature profiles. It also allows automatic control over the maintenance of nutrient level within a narrow range, pH values, dissolved oxygen and temperature values.
Objectives – To isolate and purify the green fluorescent protein.
Introduction Stage 1 Isolation – Bacteria cells need to be lysed in order to release the green fluorescent protein (GFP). – 10mL of the culture broth is collected and centrifuged at 10,000rpm for about 5 minutes to separate the cells, which will then form a pellet at the bottom of the tube.
We will now look at the 3 main methods of cell disruption.
1. Using Enzymes - The pellet is resuspended in 500 μL of TE buffer of pH 7.5 using a micropipettor. - 2 drops of lysosomes were added to the resuspended cell pellet. The enzymatic digestion of the bacteria’s cell wall will start and was left for further digestion for about 15 minutes.
2. Freezing and Thawing The tube was then placed in liquid nitrogen to freeze the content in it then was thawed in warm water. The freezing and thawing step was repeated for another 2 times to rupture the cell wall.
Bern & Milan are having fun with our poor samples :P 3. Sonication So what is sonication? Sonication is an act of applying sound (usually ultrasound) energy to agitate particles in a sample, for various purposes. In this process, the bacteria cell wall will be imploded by the pressure. This process is performed on ice for 4 cycles of 25 seconds, (giving about 10sec intervals for each cycle). Then the cells are centrifuged at 10,000 rpm for 20 minutes and the supernatant and pellet were separated out. Our KangYang has the honour :) The pellet was then resuspended in 400 μL of TE buffer. The GFP is now in the supernatant and we can view it using a fluorescent light. Stage 2: Purification Purification will be performed by using gel permeation or size exclusion chromatography. A column of a polymer gel resin is needed. The larger molecules will flow through the column at a faster rate than the small particles. Basically these molecules are separated by size! Procedures 1. Eight test tubes were labelled including a blank 2. The blank was filled with 2.0ml of ammonium bicarbonate. 3. The column was drained into a waste baker until the buffer was just even with the top of the gel bed. 4. Cell-free extract was transferred to the top of the gel bed 5. The buffer(eluent) was then collected to the 2cm mark in all test tubes 6. The sample was allowed to flow and the flow rate was adjusted to 1 drop/ 2sec interval. 7. 50 mM of ammonium bicarbonate buffer was added to the top of the column while the fractions were collected 8. Lastly, 2 ml fractions were taken from all 8 tubes 9. Absorbance values of samples taken from the various fractions were then read, using the spectrophotometer, at 476nm. Results o Absorbance readings o Graph of OD476 against fraction number Discussion In Day 4’s experiment, we had to lyse the cells to get the green fluorescent protein. We need to so this because the GFP is an intracellular protein. Which mean that the protein would not be secreted out of the cell after synthesis of the GFP. Into total, we used three techniques. Even though 1 would be enough, we did all three for practice. To start off with, we must first isolate the cells. The method of which is centrifugation. The cells were centrifuged at 10, 000 rpm for 5 minutes. The centrifugation would use gravitational force to pull down all the cells in suspension into a pellet at the end of the tube, leaving the liquid broth without the GFP at the top. This way, we can get only the cells by getting rid of the supernatant which is the broth. So, we start off with the first method of cell lysing. It is the use of enzymes. After the cell pellet was resuspended in TE buffer, we added in lysozyme, and incubate for 15 minutes. The lysozyme is an enzyme that would lyse or kill the cells. They break open the cell wall and membrane, and all the contents in the cells would be released into the environment in the tube. This would include the genetic material and etc. Also this would include the GFP that we want. A second method we used would be the freezing and thawing method. This method would require us to put the tube in liquid nitrogen, which is -120°C to freeze the cells. Then, we put the cells into warm water to thaw. We did this repeatedly for 3 times. This would cause mechanical stress to the cell wall, as the cell’s water content contract as it freezes, and expand when it is thawed. This would cause the cell wall to give way and break open the cell. The last method used was sonication. This is to implode the bacterial cell wall through vibration. This is achieved by ultrasonic waves. The waves create a vibration pressure that causes the cell wall to implode. After the cell disruption, the tube was centrifuged again to allow all the cell fragments to be pelleted, and the GFP which would be released by the cells would become suspended in the supernatant. The next thing that was done was purification. This is to get the GFP from the supernatant that we have, as they may still contain impurities. Therefore, we use gel permeation or size exclusion chromatography or gel filtration to get the green fluorescent protein. For this experiment, we used a column of polymer gel resins called the Sephadex G75. These beads contain small pores which allow molecules that are small enough to enter. Therefore, when the sample with the GFP is poured into the column, the larger molecules would flow through the column faster, while the smaller molecules interact with the resins, resulting in slower elution. The pores in the Sephadex G75 are only big enough for the GFP to diffuse in, and therefore all the other molecules in the solution would be eluted faster. This helps us to separate the GFP and the other molecules according to size. The elution was collected in fractions of 2ml. Collecting the elution in fractions of 2ml. After the purification step, the last thing to do was to analyze the results. This was done by spetrophotometry at the absorbance of 476 nm. GFP absorbs this wavelength strongly, and gives out its usual fluorescence. After this was done, a graph of fractions against absorbance was plotted. From the graph, we can see that the absorbance increases and then decrease down the fractions. This is because, at the start, the larger molecules in the solution sample were eluted, and the GFP was interacting with the Sephadex resins. Therefore, these fractions do not contain much GFP. However, as the fraction number increases, more GFP is being eluted as they are done diffusing through the resins. Then, the amount of GFP decreases again, as there is lesser and lesser GFP left in the column, and then no more is left. Questions 1. GFP has a Mr (molecular weight) around 27,000kD. 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 50,000kD would elute in a fraction before or after GFP? Why or why not? – Since the protein has a Mr of 50,000kD, which is apparently heavier than the GFP, it will not be able to trap in the fraction and also heavy particles (the protein) has less overall volume to cross over the length of the column which allows them to be eluted out before GFP. Normally, the rate of elution depends strongly on the size of particles. _ _ _ _ _LiFe of a Shr00m >.<
3. Sonication So what is sonication? Sonication is an act of applying sound (usually ultrasound) energy to agitate particles in a sample, for various purposes. In this process, the bacteria cell wall will be imploded by the pressure. This process is performed on ice for 4 cycles of 25 seconds, (giving about 10sec intervals for each cycle). Then the cells are centrifuged at 10,000 rpm for 20 minutes and the supernatant and pellet were separated out.
Our KangYang has the honour :)
The pellet was then resuspended in 400 μL of TE buffer. The GFP is now in the supernatant and we can view it using a fluorescent light.
Stage 2: Purification Purification will be performed by using gel permeation or size exclusion chromatography. A column of a polymer gel resin is needed. The larger molecules will flow through the column at a faster rate than the small particles. Basically these molecules are separated by size!
Procedures 1. Eight test tubes were labelled including a blank 2. The blank was filled with 2.0ml of ammonium bicarbonate. 3. The column was drained into a waste baker until the buffer was just even with the top of the gel bed. 4. Cell-free extract was transferred to the top of the gel bed 5. The buffer(eluent) was then collected to the 2cm mark in all test tubes 6. The sample was allowed to flow and the flow rate was adjusted to 1 drop/ 2sec interval. 7. 50 mM of ammonium bicarbonate buffer was added to the top of the column while the fractions were collected 8. Lastly, 2 ml fractions were taken from all 8 tubes 9. Absorbance values of samples taken from the various fractions were then read, using the spectrophotometer, at 476nm.
Results o Absorbance readings
o Graph of OD476 against fraction number
In Day 4’s experiment, we had to lyse the cells to get the green fluorescent protein. We need to so this because the GFP is an intracellular protein. Which mean that the protein would not be secreted out of the cell after synthesis of the GFP. Into total, we used three techniques. Even though 1 would be enough, we did all three for practice.
To start off with, we must first isolate the cells. The method of which is centrifugation. The cells were centrifuged at 10, 000 rpm for 5 minutes. The centrifugation would use gravitational force to pull down all the cells in suspension into a pellet at the end of the tube, leaving the liquid broth without the GFP at the top. This way, we can get only the cells by getting rid of the supernatant which is the broth.
So, we start off with the first method of cell lysing. It is the use of enzymes. After the cell pellet was resuspended in TE buffer, we added in lysozyme, and incubate for 15 minutes. The lysozyme is an enzyme that would lyse or kill the cells. They break open the cell wall and membrane, and all the contents in the cells would be released into the environment in the tube. This would include the genetic material and etc. Also this would include the GFP that we want.
A second method we used would be the freezing and thawing method. This method would require us to put the tube in liquid nitrogen, which is -120°C to freeze the cells. Then, we put the cells into warm water to thaw. We did this repeatedly for 3 times. This would cause mechanical stress to the cell wall, as the cell’s water content contract as it freezes, and expand when it is thawed. This would cause the cell wall to give way and break open the cell.
The last method used was sonication. This is to implode the bacterial cell wall through vibration. This is achieved by ultrasonic waves. The waves create a vibration pressure that causes the cell wall to implode.
After the cell disruption, the tube was centrifuged again to allow all the cell fragments to be pelleted, and the GFP which would be released by the cells would become suspended in the supernatant.
The next thing that was done was purification. This is to get the GFP from the supernatant that we have, as they may still contain impurities. Therefore, we use gel permeation or size exclusion chromatography or gel filtration to get the green fluorescent protein. For this experiment, we used a column of polymer gel resins called the Sephadex G75. These beads contain small pores which allow molecules that are small enough to enter. Therefore, when the sample with the GFP is poured into the column, the larger molecules would flow through the column faster, while the smaller molecules interact with the resins, resulting in slower elution. The pores in the Sephadex G75 are only big enough for the GFP to diffuse in, and therefore all the other molecules in the solution would be eluted faster. This helps us to separate the GFP and the other molecules according to size. The elution was collected in fractions of 2ml.
Collecting the elution in fractions of 2ml. After the purification step, the last thing to do was to analyze the results. This was done by spetrophotometry at the absorbance of 476 nm. GFP absorbs this wavelength strongly, and gives out its usual fluorescence. After this was done, a graph of fractions against absorbance was plotted. From the graph, we can see that the absorbance increases and then decrease down the fractions. This is because, at the start, the larger molecules in the solution sample were eluted, and the GFP was interacting with the Sephadex resins. Therefore, these fractions do not contain much GFP. However, as the fraction number increases, more GFP is being eluted as they are done diffusing through the resins. Then, the amount of GFP decreases again, as there is lesser and lesser GFP left in the column, and then no more is left. Questions 1. GFP has a Mr (molecular weight) around 27,000kD. 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 50,000kD would elute in a fraction before or after GFP? Why or why not? – Since the protein has a Mr of 50,000kD, which is apparently heavier than the GFP, it will not be able to trap in the fraction and also heavy particles (the protein) has less overall volume to cross over the length of the column which allows them to be eluted out before GFP. Normally, the rate of elution depends strongly on the size of particles. _ _ _ _ _LiFe of a Shr00m >.<
After the purification step, the last thing to do was to analyze the results. This was done by spetrophotometry at the absorbance of 476 nm. GFP absorbs this wavelength strongly, and gives out its usual fluorescence. After this was done, a graph of fractions against absorbance was plotted. From the graph, we can see that the absorbance increases and then decrease down the fractions. This is because, at the start, the larger molecules in the solution sample were eluted, and the GFP was interacting with the Sephadex resins. Therefore, these fractions do not contain much GFP. However, as the fraction number increases, more GFP is being eluted as they are done diffusing through the resins. Then, the amount of GFP decreases again, as there is lesser and lesser GFP left in the column, and then no more is left.
Questions 1. GFP has a Mr (molecular weight) around 27,000kD. 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 50,000kD would elute in a fraction before or after GFP? Why or why not?
– Since the protein has a Mr of 50,000kD, which is apparently heavier than the GFP, it will not be able to trap in the fraction and also heavy particles (the protein) has less overall volume to cross over the length of the column which allows them to be eluted out before GFP. Normally, the rate of elution depends strongly on the size of particles.
Fermentation is a process whereby cells convert substrate into useful products. To prepare for fermentation, the media, equipments and cells need to be prepared first.
The media used must contain the necessary ingredients to ensure proper cell growth for making the desired product. Before fermentation, the organism needs to be cultured or be induced to produce desired product during the growth phase. The microorganism used has desirable characteristics such as rapid growth, genetic stability and large cell size for easy removal from culture fluid.
After a suitable microorganism has been identified, the fermentation conditions have to be set up to ensure that the cells sustain its growth and produce the desired product. Parameters such as aeration, mixing, oxygen, pH, off-gas concentrations and substrate concentrations are the important factors to be controlled to obtain high yield of product.
Since Green fluorescent protein has a broad use in almost all organisms and has been used as reporter gene, cell marker, fusion tag and other uses for protein and cellular study, the need of increasing its yield has become desirable. Thus, with large scale fermentation, the green fluorescent protein can be obtained in large quantities in a short period of time instead of isolating small amounts of GFP from the jellyfish Aequorea victoria.
Experiments (Day 1 - 4) Objectives
Designer: SherMaiNe
Photographer: Kang Yang & Shermaine
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