Team:Valencia/Project/Results

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[[Team:Valencia|<font color="#047DB5">Home</font>]]
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== Achievements ==
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&nbsp;&nbsp;|&nbsp;&nbsp;[[Team:Valencia/Team|<font color="#047DB5">The Team</font>]]
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&nbsp;&nbsp;|&nbsp;&nbsp;[[Team:Valencia/Project|<font color="#047DB5">The Project</font>]]
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&nbsp;&nbsp;|&nbsp;&nbsp;[[Team:Valencia/Parts|<font color="#047DB5">Parts Submitted to the Registry</font>]]
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&nbsp;&nbsp;|&nbsp;&nbsp;[[Team:Valencia/Modeling|<font color="#047DB5">Modeling</font>]]
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&nbsp;&nbsp;|&nbsp;&nbsp;[[Team:Valencia/Notebook|<font color="#047DB5">Notebook</font>]]
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[[Team:Valencia/Project|<font color="#047DB5">Introduction</font>]]
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&nbsp;&nbsp;|&nbsp;&nbsp;[[Team:Valencia/Project/Objectives|<font color="#047DB5">Objectives</font>]]
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&nbsp;&nbsp;|&nbsp;&nbsp;[[Team:Valencia/Project/Results|<font color="#047DB5">Results</font>]]
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&nbsp;&nbsp;|&nbsp;&nbsp;[[Team:Valencia/Project/Modeling|<font color="#047DB5">Modeling</font>]]
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<h2>1.- Construction of a ''Liquid Culture Calorimeter'' (LCC).</h2>
 
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<ol><li>Developed a brand new instrument: [[Team:Valencia/Project/Lab_work | '''LCC (Liquid Culture Calorimeter)''']] for real-time, high precision measurement of the internal temperature of a thermically isolated liquid culture. This instrument is a new technical standard that supports the characterization of other temperature-related BioBricks Parts or Devices. <br><br></li>
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<li>Demonstrated, for the first time, that UCP-1 (termogenin) is, as expected, [[Team:Valencia/Project/Lab_work/2_experiments | '''able to produce a significant increase in temperature''']] in recombinant UCP-expressing yeasts. <br><br></li>
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<li>Characterized [[Team:Valencia/Parts | '''four new biobricks''']] (a UCP-expressing yeast, a control strain and two mutant strains with enhanced uncoupling activity) through temperature measurements with the LCC, as well as from relative growth data of the yeast cultures. <br><br></li>
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<li>Outlined a new approach to Human Practices in Synthetic Biology by writing a [[Team:Valencia/Project/Ethics|'''Proposal for a Code''']], that includes a novel concept: concentric ethical units, that might be very useful not only for the Hot Yeast Project but also for any Synthetic Biology research. <br><br></li>
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<li>Developed an [[Team:Valencia/Project/Modeling | '''effective model''']] to characterize the kinetic behavior of thermogenin. This model describes UCP1 functionality in terms of heat production. In future, the obtained values for the parameters can be used by other groups which intend to incorporate this part in their constructions.<br><br></li>
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<li>Provided help to the community as much as our knowledge permitted. Bearing in mind that this was our third participation on the iGEM competition, we felt that it was our responsibility to help other teams as much as we could. At beginning of September, we mailed the team of [[Team:Bologna|'''Bologna''']], [[Team:EPF-Lausanne|'''EPF-Lausanne''']] and [[Team:Paris|'''Paris''']] regarding a fluorescent phenomenon known as [http://en.wikipedia.org/wiki/Fluorescence_resonance_energy_transfer '''FRET'''] that could give problems in experimental results interpretation. We knew of that phenomenon from our project in [https://2007.igem.org/Valencia '''iGEM 2007'''], and since then are very cautious in designing systems that have two fluorescent proteins on the same cell.</li>
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Our LCC consists of four commercial plastic and glass thermo flasks which we modified. We cut off the handle and drilled a hole in the cap. We inserted the thermocouple through the hole and covered the whole calorimeter with insulation foam called Armaflex.
 
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[[Image:Valencia_LCC.png|450 px|right]]
 
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[[Image:Valencia_yeasty2.png|300px]]
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The thermocouples where connected to a data logger that stores temperature evolution during the experiment. At the same time, this data logger was connected to the computer. We developed a program which interprets the data and shows an updated line chart every 30 seconds.
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<h3>Characterization results</h3>
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Further work:
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After trying different combinations of revolution speed, flask tilt and liquid volume, we obtained a combination of conditions suitable for the experiment: 150 rpm, approximately 10 º tilt.
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<ol>Regulation of the thermogenin-associated thermal increase could be performed using heat and/or cold shock protein promoters. These promoters, controlling genes responsible of activation or inhibition of thermogenin or another protein involved in the proton gradient might be used to mantain the temperature of the cultures within a given range. </ol>
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<h3>Troubleshooting</h3>
 
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We have encountered some adversities regarding Armaflex. This material is sensitive to mechanical damage and especially to water. No matter how careful we were, we found the insulation capacity of our system had drastically decreased in the first weeks.
 
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|[[Image:Valencia_LCC4.JPG|250px|left]]
 
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|The main problem was cleaning the LCCs, since it was very difficult for them not to get wet. We tried different protocols, such as covering them with aluminum foil and sealing with parafilm or using funnels, but the water got inside anyway. Every time the temperatures were falling more than usual, we had to stop the experiment, remove Armaflex cover and use a hair dryer to dry it.
 
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Covering the outside was not enough; we needed something that covered the Armaflex in the inside, between the flask and the Armaflex. We used a garbage bag to do this, and surprisingly it has worked up to now. 
 
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|With regard to the LCCs characterization, we have also gone through different conditions combinations. The first problem we encountered was the apparition of sudden periodic oscillations of temperature in some of the experiments. The best explanation was that water was somehow coming in contact with the thermocouple, which was placed in the upper part of the flask at that time. To try and solve that, we reduced the liquid volume, but the problem persisted. After watching this phenomenon was not produced when the shaking was stopped, it became clear the oscillations were related to it, probably because of a condensation process.
 
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The solution to this problem appeared, as usual, when we were not looking for it. In order to provide more oxygen to the culture, we increased the shaking speed and the flask tilt in an experiment with water. Surprisingly, the periodic oscillations disappear and the temperature not only did not fall so much, but slightly increase. We found we could regulate the temperature with these two factors; therefore, we adjusted them so as to obtain almost stable temperature with a high shaking speed suitable for culture growth.
 
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<center><font face="trebuchet ms" style="color:#047DB5" size="3"> [[Team:Valencia/Project/Modeling | <font color="#047DB5">'''3.Modeling'''</font>]] << '''Results ''' >> [[Team:Valencia/Project/Ethics| <font color="#047DB5">'''Ethics'''</font>]]</font>
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Another condition we changed was the position of the thermocouple inside the flask. They had been always placed in the upper part, having no contact with the liquid because of contamination reasons. However, our engineer team members suggested it would be better for the thermocouples to be submerged in the liquid. After carrying out an experiment, we saw there was not too much different between both positions, but there were not contaminations problems either. Since the data obtained is more exact with the thermocouple submerged, we decided to keep them that way.
 
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Latest revision as of 22:20, 29 October 2008



Achievements

We have:

  1. Developed a brand new instrument: LCC (Liquid Culture Calorimeter) for real-time, high precision measurement of the internal temperature of a thermically isolated liquid culture. This instrument is a new technical standard that supports the characterization of other temperature-related BioBricks Parts or Devices.

  2. Demonstrated, for the first time, that UCP-1 (termogenin) is, as expected, able to produce a significant increase in temperature in recombinant UCP-expressing yeasts.

  3. Characterized four new biobricks (a UCP-expressing yeast, a control strain and two mutant strains with enhanced uncoupling activity) through temperature measurements with the LCC, as well as from relative growth data of the yeast cultures.

  4. Outlined a new approach to Human Practices in Synthetic Biology by writing a Proposal for a Code, that includes a novel concept: concentric ethical units, that might be very useful not only for the Hot Yeast Project but also for any Synthetic Biology research.

  5. Developed an effective model to characterize the kinetic behavior of thermogenin. This model describes UCP1 functionality in terms of heat production. In future, the obtained values for the parameters can be used by other groups which intend to incorporate this part in their constructions.

  6. Provided help to the community as much as our knowledge permitted. Bearing in mind that this was our third participation on the iGEM competition, we felt that it was our responsibility to help other teams as much as we could. At beginning of September, we mailed the team of Bologna, EPF-Lausanne and Paris regarding a fluorescent phenomenon known as [http://en.wikipedia.org/wiki/Fluorescence_resonance_energy_transfer FRET] that could give problems in experimental results interpretation. We knew of that phenomenon from our project in iGEM 2007, and since then are very cautious in designing systems that have two fluorescent proteins on the same cell.
      

Valencia yeasty2.png


Further work:

    Regulation of the thermogenin-associated thermal increase could be performed using heat and/or cold shock protein promoters. These promoters, controlling genes responsible of activation or inhibition of thermogenin or another protein involved in the proton gradient might be used to mantain the temperature of the cultures within a given range.



3.Modeling << Results >> Ethics


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