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Cells (in our case E. coli) are delicate and require a great deal of attention to detail for them to grow well. A slight change to their surroundings can vastly affect their ability to reproduce and generate products.
Oxygen is required for most microbes to live and grow, however if in excess, it is toxic! The high oxygen generates free radicals, high levels of free radicals affects their cell growth and may even kill them. However oxygen also has a low solubility in water which makes it necessary for us to introduce methods which will allow for enough oxygen to dissolve and meet the cell oxygen demand.
For most cells, the optimal pH is around 6.5 to 7.0, any more or less then that places stress on the cells.
Lastly, temperature is best maintained around 37 degrees Celsius as most cells grow best. Anything above a temperature of 40˚C will affect the cells, and most likely killing them since proteins will be denatured in high temperature. It is also important that the fluctuations in the temperature are kept to a minimal as a constantly changing environment is defiantly stressful for us, much more so for cells.
2. Describe the principle of the spectrophotometer which was used to determine the cell density (OD600). Why was 600nm used?
A spectrophotometer works by measuring the amout of light, sent out by a light source, received by the detector. The light source sends out light which are split up by a prism into various bands of differing wavelengths. An appropriate slit or device then allows the specific band of wavelength through and shines it on the sample or blank.
The amount of light received by the detector is then computed and translated into a value kown as transmittance or absorbance. Cells absorbs light maximally at a wavelength of 600nm hence that wave length is used.
3. Is GFP a primary or secondary metabolite? At which phase should the product be harvested? At which phase was the product actually harvested?
GFP is a secondary metabolite product. This product is generated during the late growth phase and throughout the stationary phase. Therefore the product should be harvested during the late stationary phase to get the maximum yield.
4. What are some advantages of using a computer control system? From the history chart, comment on the effectiveness of the computer control.
Advantages include:
There are four sections to Day 3:
Part I (Procedures)
Part II (Growth Graph)
Part III (History Plot)
You are Here: Part IV (Futher Questions)
(click to view enlarged image)Since it is said that both the pO2 level and the stirrer speed are inversely proportional, we shall explain the non-coordinated graph of pO2 and stirrer speed between time 2.5h to 5h.
As observed, the stirrer speed is increasing rather consistently while the pO2 level remains constant. The reason being that this is the log phase.
During the log phase O2 demand level increases without the stirrer the pO2 level will drop consistently, however the automated system deems that the lower limit for pO2 level is 24% saturation. Therefore, to keep the pO2 at least at 24% saturation, the stirrer speed increases to make the dissolving rate of O2 equivalent to the O2 uptake rate. As the uptake rate and the dissolving rate is the same, the net change is zero. Hence the pO2 level remains constant during this period.
There are four sections to Day 3:
Part I (Procedures)
Part II (Growth Graph)
You are Here: Part III (History Plot)
Part IV (Futher Questions)
Discussion for Cell Growth
By plotting the cell growth curve, we can actually determine the:
1) Different stages of E.coli cell growth (lag, log, stationary, death)
2) Duration of the growth stages
3) Specific growth rate at a specific timing
To plot the curve, it is necessary to determine the cell concentration. On the other hand, to determine the cell concentration, the absorbance of the samples at every hour must be measured using a laboratory equipment known as the spectrophotometer ( refer to further questions for the principle of the machine).
Samples were extracted from the bioreactor 10 hours at every hour from 9am to 6pm (excluding the control sample) and the absorbance was taken.
The Beer-Lambert Law states that the absorbance of a material in solution is directly dependent on the concentration of that material.A α c The material in this case is the cell concentration (x).
With the absorbance values, we can deduce the cell concentrations from the following steps:
By comparing the two equations above, we can see that the efficient coefficient (ε) and the path length of the cuvette (l) are identical. Thus, the two equations can be combined into the equation below and the ε and l can be eliminated.
As a result, the difference in absorbance values obtained is identical to the difference cell concentration values at each respective hour.
From the cell concentration values, the values of Log (c/c0) or Log (x/x0) that we are required to find can be calculated so that the cell growth graph can be plotted.
Samples were extracted from the bioreactor for 10 hours at every hour from 9am (first hour) to 6pm (last hour).
From the graph plotted, only the exponential and the stationary phases could be observed. However, the lag phase and the death phase could not be seen and were not known. The slope of the exponential phase = μmax, which is the maximum cell growth rate. The slope of other parts of the curve = μ, which is the specific cell growth rate.
Phase (1): The lag phase
For the absence of the lag phase observed, one possible reason to support the observation would be due to the fact that the absorbance of the first fraction taken was right after the lag phase. Therefore the lag phase was not captured in this graph.
However, the lag phase can be deduced from the history plot (refer to the next page History Plot) which is 0.8 hours from the start (9am instead of 8.30am from the history plot).
Phase (2): The exponential phase
As a result of the deduction, the exponential phase started at 0.8 hours until the 5th hour whereby graph rose steadily after lag phase. This was logarithmic phase, the period where the cells were expected to grow exponentially. This phase happened after the cells had adapted to the environment already. Hence, they started to grow and divided at the maximum rate.
Phase (3): The stationary phase
Stationary phase, also called the idiophase, happened when cell growth was equal to cell death. This occurs at the 5th hour to the 17.5th hour (observed from the history plot) on the graph whereby the slope equals to zero. This phase occurred because the rate of cell growth is more or less equal to the rate of cell death, and can be triggered by the exhaustion of a particular critical nutrient essential for maintaining cell growth, or the accumulation of toxic metabolic products.
Phase (4): The death phase
Finally, the death phase was not detected and could not be observed from the graph. It refers to the period of exponential decay and it occurs either due to the depletion of cellular reserves of energy or accumulation of toxic waste products.
Please refer to the History Plot for further details!
There are four sections to Day 3:
Part I (Procedures)
You are Here: Part II (Growth Graph)
Part III (History Plot)
Part IV (Futher Questions)
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1. The ampicillin and arabinose stock solution had been prepared before hand and filtered and sterilized.
2. The medium was cooled to below 50°C. Ampicillin was added until it reached a final concentration of 100μg/ml, while arabinose was added until it reached a final concentration of 0.2%.
3. The following parameters were set up:
4. The fermentor was inoculated with 100ml of seed culture (5% of fermentation medium volume).
5. The fermentation was then continued for another 24 hours.
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2. The pH probe, pO2 probe, foam and level probe were installed into the top plate. Necessary adjustment of the height for the foam and level probe is carried out when necessary.
3. The addition agent lines for acid, base and antifoam were connected and the levels in the storage bottles were checked.
4. The exhaust condenser, air inlet and exhaust filters and manual sampler unit were installed.
Further questions...
(a) What is meant by calibration of the pH probe?
Calibration of the pH probe is meant to set the pH point to be uniformed within the fermenter. Hence, a slight change in pH can be detected by the pH probe.
(b) Why is hydrochloric acid (HCl) not suitable as a correction agent for pH?
(2) For the sterilization of the bioreactor:
2. All silicone tubings except for exhaust filter and female STT coupling of sampling unit were clamped. All filters and sockets were covered with aluminium foil to protect from condensing moisture.
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3. The bioreactor was autoclaved with steam at 121 degree celsius for 20 minutes.
4. The pO2 electrode was polarized for at least 6 hours and calibrated by aerating with nitrogen.
5. The addition lines were connected to peristaltic pumps.
1. Luria-Bertani (LB) medium was used for both seed-culture and fermentation media.
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2. 1000ml of distilled water was added to 25g LB-powder containing bacto-tryptone, yeast extract and sodium chloride (NaCl).
3. The pH was pre-equilibrated in the powder; therefore, no equilibration was needed.
4. The media was autoclaved at 121 degree Celsius for 20 minutes.
5. Ampicillin was added to the final concentration of the media once the broth had cooled to below 50 degree Celsius.
6. The media was stored at 4 degree Celsius till inoculation.
Further questions...
(a) Bacto-tryptone
(b) Yeast extract
It provides any type of organic molecule that a growing bacteria would need and essential for cellular metabolism. Glutamate contributes as an intermediate in processes such as glycolysis, gluconeogenesis and citric acid cycle. Vitamins are bio-molecules that act as catalysts and substrates in chemical reaction.
(c) NaCl
(d) dH2O
(e) pH
(ii) What is the purpose of ampicillin?
(iii) Why is ampicillin added only after autoclaving?
(4) For the preparation of seed culture:
1. The pGLO transformed E. coli was streaked onto a LB/Amp (ampicillin)/Ara (Arabino) plate and was incubated for 24 hours.
2.After incubation, several colonies were obtained and transferred to the flask containing 100ml LB medium with ampicillin.
3. The flask was left in shaking incubator and incubated at 32 degree Celsius for 24 hours or at r.t.p for 48 hours. This is to facilitate the purpose of scaling-up.
Further questions...
(a) What is the purpose of arabinose?
(b) Describe the sterile techniques used in seed preparation.
(c) Why do we perform step-wise scale-up instead of transforming directly to the fermenter?
A photo of Our Savior: the beloved bioreactor
Our Savior has many different important parts that serve different functions. Long live the bioreactor!
1. Motor - to drive the motion of the impeller by supplying electrical power
2. Impeller - to mix the media such that cells are prevented from settling at the bottom of the fermenter, and to distribute the bubbles evenly throughout the broth
3. Sparger - to supply sufficient oxygen and other essential gases required by sparging in air
4. Baffles - to ensure even gas and nutrient distribution, such that there is good mixing
5. Inlet air filter - to sterilize the inlet air stream and remove any possible contaminants using a 0.22nm filter
6. Exhaust air inlet - to sterilize the outlet air stream by also using filter
7. Rotameter - to measure the gas flow rate
8. Pressure gauge - to measure air pressure in the fermenter
9. Temperature probe - to measure the temperature of the media
10. Cooling jacket - to cool or heat the water
11. pH probe - to measure pH of media
12. Dissolved oxygen probe - to measure the amount of dissolved oxygen in the broth and ensure cells have obtained sufficient oxygen and other essential gas components
13. Level probe - to measure the level of the liquid in the bioreactor
14. Foam probe - to detect increasing levels of foam that may form during the run, such that the appropriate action can be taken
15. Acid / Base - to neutralise the changes incurred during the fermentation process
16. Antifoam - to prevent build-up of foam and break the surface tension to break bubbles that may coagulate during the run
17. Sampling tubes - for sampling of cultures at consistent intervals in order to monitor the growth of cells and their constituent products
18. Control panel - to settle parameters
Our blog would be in shambles if it weren't for some help from these great sources! Lots and lots of arigatou to:
For pictures within The Secrets behind GFP section:
Figure 1.1:
http://www.ens-lyon.fr/RELIE/PCR/ressources/apects_techniques/tp_gfp/GFPuv1.htm
Figure 1.2:
http://www.biologicalprocedures.com/bpo/arts/1/40/m40f1lg.gif
Figure 1.4:
http://www.sct.ub.es:802/Tutorial/imagenes/1EMBx500negropeq.gif
Figure 1.5:
http://fireflyforest.net/images/fireflies/2006/November/fluorescent-scorpion.jpg
Figure 1.6:
http://www.mshri.on.ca/nagy/graphics/GFP%20mice.jpg
Figure 1.7:
http://www.flickr.com/photos/adrianv/348308324/
Figure 1.8: (for the Pokémon picture!)
http://upload.wikimedia.org/wikipedia/it/3/39/072_Tentacool.png
For the information within The Secrets behind GFP section:
http://userpages.umbc.edu/~jili/ench772/intro.html
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
http://en.wikipedia.org/wiki/Green_fluorescent_protein
For answers to questions:
(Day 1 Experiment 1)
http://www.microbialcellfactories.com/content/5/1/21
(Day 1 Experiment 2)
http://www.newton.dep.anl.gov/askasci/bio99/bio99369.htm
http://www.bio-rad.com/LifeScience/pdf/Bulletin_9565.pdf
(Day 3 Experiment 3)
http://www.chm.davidson.edu/java/spec/spec.html
Fig. 1.1
Fig. 1.2
Section 1.2: History of GFP
The timeline below depicts the history and the discovery of GFP, and the brilliant scientists who contributed to its discovery. Click on the link below for a larger and clearer version:
Fig 1.3
Click here for a larger and clearer diagram of the timeline.
Section 1.3: Structure of GFP
GFP has a unique can-like shape consisting of an 11-strand β-barrel with a single alpha helical strand containing the fluorophore running through the center. This protein contains 238 amino acids. Below is the beautiful structure of GFP... behold!
Fig. 1.4
Fig 1.5: a scorpion fluorescing under UV light
Fig 1.6: a cool animation comparing a genetically-engineered rat glowing with the help of GFP, and a normal wild-type
Figure 1.7 shows "Alba" the green fluorescent bunny
Section 1.5: Incorporation of GFP into Escherichia coli cells
The diagram below summarizes the procedure. Click on the image for an enlarged version.
Figure 1.8
1. Aequorea Victoria cells were broken down to obtain genomic DNA that contain genes encoding for GFP.
2. pGLO plasmid vectors are isolated from E.coli. pGLO plasmid vector contains two genes for selection, one is the ampicillin resistance gene which encodes for B-lactamase and another was the gene encodes for the regulation by the carbohydrate arabinose.
3. The released genomic DNA and pGLO plasmid vectors were then cleaved using the same restriction enzyme.
4. The cut genomic DNA and pGLO plasmid vectors were stitched together by using ligase enzyme.
5. The E. coli cells were being transformed by recombinant vector with gene of interest after ligation step.
6. Only the transformed E.coli cells would then be able to grow on Luria-Bertani agar plate with ampicilin and arabinose due to they contain the genes for selection. Other non-transformed cells would not grow.
7. The E. coli cells that contained gene of interest could be verified by placing under UV light. The arabinose present in the agar plate would activate the GFP gene to fluoresce under UV light.
8. Once the GFP-producing colonies were isolated, they are innoculated onto another agar plate (with same ingredients) to obtained pure cultures (enrichment method).
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