Difference between revisions of "Team:Uppsala/Design"

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   <h1>Design</h1>
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   <h1>Bioreactor</h1>
 
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   In this iGEM project we faced the need to characterize a promoter induced by the NahR protein [1], which is a regulated promoter, induced by salicylate. We decided to compare it with different amounts of salicylate concentrations and also with two constitutive promoters (the J23101 and J23110). Among the existing approaches for measuring the strength of a promoter, we chose to use fluorometry. Even though we did not have access to a fluorometer in our lab, we decided to go through with this approach while searching for a solution. One of the solutions was to build our own fluorescence measurement instrument. Hence, some of our team members decided to work around the idea of constructing a custom fluorescence measurement instrument (or fluorometer), while others searching/asking for an available fluorometer around.
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   To implement the Decyclifier where it is most needed, in the waste plants, we designed a bioreactor (see figure 1) that would be part of the waste remediation process at facilities such as Uppsala Vattenfall’s power plant. In the burning of waste, the incinerated waste is refined in three steps. The first step seperates larger metal objects and the later two refines the ashes through electrofiltration and a water-acid filtering systems. Through the process, two types of ash contain considerably higher concentrations of PAHs. We suggest that these two types of ash should be sludged with water and supplied to the Decyclifier bioreactor.
 
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  <h2>Fluorometry</h2>
 
 
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   Fluorometry is an analytical technique for identification and measurement of fluorescent compounds and targets like cells, proteins, nucleotides, or labeled targets with fluorescent tags [2]. It works by emitting light in a specific wavelength that excites electrons in the molecules of a respective compound. That causes the compound to emit light, typically within the visible spectrum. Instruments take advantage of this emitted light in order to measure the fluorescence level of the compound.
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   For the Decyclifier reactor we purpose a simple batch-mode model, due to the lengthy process of the PAH degradation (approximatly 10weeks for a 100m3 batch). With a suggested ratio of 2:1 water to ash-sludge, approximatly 33m3 of ash could be purified per batch (Li <i>et al</i>., 2002). According to Robies-González <i>et al</i>., levels of purification in similar systems reached between 70 and 98% of degradaded PAHs (González <i>et al</i>., 2008). With utilization of our biosurfactant system and the optimized activity of our modified Laccase, one might assume that the Decyclifier reactor should be able to reach atleast the higher level of purification, possibly also in shorter amount of time. There is however no way to say for certain without testing the system under proper running conditions.
 
   </p>
 
   </p>
 
   <p>
 
   <p>
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   Fluorometry is an analytical technique for identification and measurement of fluorescent compounds and targets like cells, proteins, nucleotides, or labeled targets with fluorescent tags [2]. It works by emitting light in a specific wavelength that excites electrons in the molecules of a respective compound. That causes the compound to emit light, typically within the visible spectrum. Instruments take advantage of this emitted light in order to measure the fluorescence level of the compound.
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   Based on similar reactors the innoculating concentration of the Decyclifier in the reactor should be about 50 g/kg. As such it will be nessecary to also include a smaler pilot reactor for growth of the inoculating culture prior to PAH degredation. Preferably this reactor should be run using similar conditions as the main reactor to make sure that the cells are indeed functionaling properly.
 
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  <h2>Custom Fluorometer</h2>
 
 
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   Since there are a lot of instruments that can measure fluorescence, we had to choose one that suits our case. Depended on our low budget, we had to choose the best combination of a cheaper and easier approach, which as we concluded is the filter fluorometer. This type of fluorometer uses optical filters in order to let only the desirable wavelengths to pass, while blocking the rest of the spectrum [3].
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   Judging from our labtests, the Decyclifier is capable of surviving high concentrations of pure napththalene and can thus be assumed to also be able to survive fairly high concentrations of PAHs in the toxic ash. A key issue however is the fact that ash contains many other toxic substances in addition to PAHs. As of yet no tests regarding the cells viability in direct contact with the ash has been performed. Lab tests were planned to assert how well the Decyclifier would survive in cultures with actuall ash, however unfortunatly Vattenfall were unable to supply us with the ash in time. This would of course be a key experiment for the future assessment of the Decyclifier system.
 
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   Originally, filter fluorometers have one excitation filter between the light source and the sample, in order to let pass only the wavelength that excites the compound. Also, they have an emission filter in order to let only the wavelength emitted by the compound to pass out to a light sensor. The actual measurement is performed by the light sensor that captures the light’s intensity, that comes out of the emission filter. Based on that and taking advantage of existed materials, we had as a base of our fluorometer project the Arduino microcontroller [4]. The rest of the main fluorometer components are two green LEDs used as light source, one longpass optical filter at 590nm (FGL590) used as an emission filter, and a light sensor (TSL250R) that outputs an analog signal, with the voltage being directly proportional to the incoming light intensity. All of those components are attached in a 3D printed cuvette holder chamber (designed by Dr. Erik Gullberg), made specifically for this purpose.
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   Due to the relative toxicity provided through other components of the ash, it is important to remember that the remaining material still needs to be disposed of after the reactor run time has finished. As such the Decyclifier reactor will need to be emptied and the remaining slugde deposited as was previously done, however now no longer containing PAHs.
 
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   Moreover, in the design of the custom fluorometer included two buttons and two extra indication LEDs. The function of the one button is to start the measurement that is about to take place, while the other button is used in order for the user to change mode. One of the available modes is the Comparison mode and the other one is the Timelapse mode. The indication LEDs are used to indicate which mode is currently running. The Comparison mode is used in order to compare several samples interchangeably, by inserting a new cuvette with a new sample each time and pressing the start button. The Timelapse mode is almost the same piece of code, with the difference that its running time and the intervals between each read are way longer than the ones in the Comparison mode. The Timelapse mode is useful when we want to measure the changes in the fluorescence intensity of one sample over time.
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   A major necessisty for a well functioning reactor is the maintenance of optimal and homeostatic growing conditions, such as temperature, pH, O<sub>2</sub> and CO<sub>2</sub>. This requires a constant rate of stirring throughout the system which can be achieved through a multiple bladed rotor originating from the top of the reactor and baffles attached to the sides. The baffles serve to direct the flow of the liquid phase in an even pattern following the edges of the container. Temperature will be regulated externally using a fluidbased coil jacket with a seperate heat regulation system coupled with an internal sensor. This would be the best method for specific and swift temperature regulation, as it is far easier to manipulate than a non-coil heat jacket system. Gas levels as well as pressure can be managed using a oxygen supplying gas input line coupled with an oxygen saturation sensor and a pressure regulated output valve. PH can be regulated in a similar fashion using a pH-electrode coupled with a bas/acid supplying system. Finally a stirred tank reactor of this size is bound to create foam and as such a proper anti-foaming agent must be added (Wang <i>et al</i>, 2008). It is also possible to introduce our flourometer into the system as a mean of meassuring the density of the cell culture. This can be achieved by attaching a smal cell to the side of the reactor, with a small but constant amount of fluid flowing through it, fitted with our devise.
 
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  Below in the Figure 1, is data gathered from the Comparison mode representing the relative fluorescence of each sample.
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  <figcaption><b>Figure 1</b>: Data gathered from the Comparison mode. We can see the correlation between the samples containing different amounts of salicylate concentration.</figcaption>
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  Finally, the fact that we use an analog light sensor means that the output values are very sensitive to external or internal noise. For this reason the circuit should be as insulated as possible and the output data should sampled and filtered. Since our fluorometer is in a prototyping phase all this period, we were not able to have a very good insulated circuit. Thus, we really had the need to have a good sampling and filtering algorithm. So, we sacrificed some speed in order to capture more samples per measurement, and we also implemented a combination of a median and a mean filter in our sampling data. The median part of the Median-­Mean filter, is modified to capture not a single median value, but several values around the median. Then, the mean part of the Median-­Mean filter calculates the average of those median values, for the final result.
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  A picture of the actual construct is presented in Figure 2.
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  <figcaption><b>Figure 2</b>: The fluorometer construct in action. At that point of time was in the prototyping phase.</figcaption>
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   <h2>References</h2>
 
   <h2>References</h2>

Revision as of 12:42, 18 September 2015



Bioreactor

To implement the Decyclifier where it is most needed, in the waste plants, we designed a bioreactor (see figure 1) that would be part of the waste remediation process at facilities such as Uppsala Vattenfall’s power plant. In the burning of waste, the incinerated waste is refined in three steps. The first step seperates larger metal objects and the later two refines the ashes through electrofiltration and a water-acid filtering systems. Through the process, two types of ash contain considerably higher concentrations of PAHs. We suggest that these two types of ash should be sludged with water and supplied to the Decyclifier bioreactor.

For the Decyclifier reactor we purpose a simple batch-mode model, due to the lengthy process of the PAH degradation (approximatly 10weeks for a 100m3 batch). With a suggested ratio of 2:1 water to ash-sludge, approximatly 33m3 of ash could be purified per batch (Li et al., 2002). According to Robies-González et al., levels of purification in similar systems reached between 70 and 98% of degradaded PAHs (González et al., 2008). With utilization of our biosurfactant system and the optimized activity of our modified Laccase, one might assume that the Decyclifier reactor should be able to reach atleast the higher level of purification, possibly also in shorter amount of time. There is however no way to say for certain without testing the system under proper running conditions.

Based on similar reactors the innoculating concentration of the Decyclifier in the reactor should be about 50 g/kg. As such it will be nessecary to also include a smaler pilot reactor for growth of the inoculating culture prior to PAH degredation. Preferably this reactor should be run using similar conditions as the main reactor to make sure that the cells are indeed functionaling properly.

Judging from our labtests, the Decyclifier is capable of surviving high concentrations of pure napththalene and can thus be assumed to also be able to survive fairly high concentrations of PAHs in the toxic ash. A key issue however is the fact that ash contains many other toxic substances in addition to PAHs. As of yet no tests regarding the cells viability in direct contact with the ash has been performed. Lab tests were planned to assert how well the Decyclifier would survive in cultures with actuall ash, however unfortunatly Vattenfall were unable to supply us with the ash in time. This would of course be a key experiment for the future assessment of the Decyclifier system.

Due to the relative toxicity provided through other components of the ash, it is important to remember that the remaining material still needs to be disposed of after the reactor run time has finished. As such the Decyclifier reactor will need to be emptied and the remaining slugde deposited as was previously done, however now no longer containing PAHs.

A major necessisty for a well functioning reactor is the maintenance of optimal and homeostatic growing conditions, such as temperature, pH, O2 and CO2. This requires a constant rate of stirring throughout the system which can be achieved through a multiple bladed rotor originating from the top of the reactor and baffles attached to the sides. The baffles serve to direct the flow of the liquid phase in an even pattern following the edges of the container. Temperature will be regulated externally using a fluidbased coil jacket with a seperate heat regulation system coupled with an internal sensor. This would be the best method for specific and swift temperature regulation, as it is far easier to manipulate than a non-coil heat jacket system. Gas levels as well as pressure can be managed using a oxygen supplying gas input line coupled with an oxygen saturation sensor and a pressure regulated output valve. PH can be regulated in a similar fashion using a pH-electrode coupled with a bas/acid supplying system. Finally a stirred tank reactor of this size is bound to create foam and as such a proper anti-foaming agent must be added (Wang et al, 2008). It is also possible to introduce our flourometer into the system as a mean of meassuring the density of the cell culture. This can be achieved by attaching a smal cell to the side of the reactor, with a small but constant amount of fluid flowing through it, fitted with our devise.

References

Schell, Mark A. "Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida." Proceedings of the National Academy of Sciences 83, no. 2 (1986): 369­373.

Curry, Robert E., Harry L. Pardue, Glen E. Mieling, and Robert E. Santini. "Design and Evaluation of a Filter Fluorometer That Incorporates a Photon­-Counting Detector." Clinical chemistry 19, no. 11 (1973): 1259-­1264.

Banzi, Massimo. Getting started with Arduino. " O'Reilly Media, Inc.", 2011.