Difference between revisions of "Team:Exeter/Collaborations"

 
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<a href="#section_1" class="banner_link col-xs-6 col-sm-3"><span class="oneline">Newcastle</span></a>
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<a href="#section_1" class="banner_link col-xs-6 col-sm-2"><span class="oneline">Newcastle</span></a>
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<a href="#section_2" class="banner_link col-xs-6 col-sm-3"><span class="oneline">Purdue</span></a>
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<a href="#section_2" class="banner_link col-xs-6 col-sm-2"><span class="oneline">Purdue</span></a>
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<a href="#section_3" class="banner_link col-xs-6 col-sm-3"><span class="oneline">Glasgow</span></a>
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<a href="#section_3" class="banner_link col-xs-6 col-sm-2"><span class="oneline">Glasgow</span></a>
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<a href="#section_4" class="banner_link col-xs-6 col-sm-3"><span class="oneline">Edinburgh</span></a>
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<a href="#section_4" class="banner_link col-xs-6 col-sm-2"><span class="oneline">Edinburgh</span></a>
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<a href="#section_5" class="banner_link col-xs-6 col-sm-2"><span class="oneline">Stanford&nbsp;Brown</span></a>
 
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                 <p id="pp">A 0.65m insulated copper wire (cross-sectional area, 0.05m^2) was passed through the centre of a 50mL falcon tube containing the liquid medium to be measured, suspended in a water bath (Fig. 1b). Three thermocouples (Pico Technology TC-08) were attached, one to the wire in contact with the insulation using blue tack, one suspended in the liquid medium 5mm from the wire, and one in the water bath. Power was supplied to the wire at 5A, 1.8V for 600s, generating a small temperature increase of approximately 2&deg;C above room temperature (23 &plusmn; 1&deg;C) to avoid convection effects. Our experimental setup was calibrated to the thermal conductivity of <a href="http://www2.bren.ucsb.edu/~dturney/WebResources_13/WaterSteamIceProperties/PropOfWaterFrom0to100Celcius.pdf">water</a> at 20&deg;C, 598.4 $\frac{mW}{Km}\text{ }$. Measurements of two growth media, liquid broth (LB) and M9 were repeated at least in quintuple.</p>
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                 <p id="pp">A 0.65m insulated copper wire (cross-sectional area, 0.05m<sup>2</sup>) was passed through the centre of a 50mL falcon tube containing the liquid medium to be measured, suspended in a water bath (Fig. 1b). Three thermocouples (Pico Technology TC-08) were attached, one to the wire in contact with the insulation using blue tack, one suspended in the liquid medium 5mm from the wire, and one in the water bath. Power was supplied to the wire at 5A, 1.8V for 600s, generating a small temperature increase of approximately 2&deg;C above room temperature (23 &plusmn; 1&deg;C) to avoid convection effects. Our experimental setup was calibrated to the thermal conductivity of <a href="http://www2.bren.ucsb.edu/~dturney/WebResources_13/WaterSteamIceProperties/PropOfWaterFrom0to100Celcius.pdf">water</a> at 20&deg;C, 598.4 $\frac{mW}{Km}\text{ }$. Measurements of two growth media, liquid broth (LB) and M9 were repeated at least in quintuple.</p>
  
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<span>Figure 1a - experimental setup of the transient hot wire method.</span>
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            <span class="caption">Fig. 1 - A picture of the experimental apparatus used for finding conductivitys.</span>
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<span>Figure 1b - arrangement of thermocouples within the falcon tube measuring thermal conductivity using the transient hot-wire method.</span>
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            <span class="caption">Fig. 2 - A close up of the experiment showing the wiring inside the falcon tube.</span>
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                <p id="pp">Nagasaka and Nagashima noted that wire insulation has negligible impact on the measurement of the thermal conductivity of saline solutions (Nagasaka and Nagashima 1981) and thus can be described by the equation: $$ \lambda = \frac{Q}{4\pi\Delta T}\ \ln{(t)}$$ where $Q$ is the power per unit length of the wire, $Q = \frac{(I \times V)}{Length}$, and $\Delta T$ is the change in temperature over time $t$, such that a linear fit of a $T$ vs. $ln(t)$ plot will yield the conductivity (Fig. 2).</p>
 
  
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                 <p id="pp">We found the thermal conductivity of LB and M9 to be similar to that of water, at 605 $\pm$ 20 $\frac{mW}{Km}\text{ }$ and 570 $\pm$ 30 $\frac{mW}{Km}\text{ }$ respectively.</p>
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<span>Figure 2 - representative plots of temperature against natural log of time for water, liquid broth and M9 media. A fit was applied from the point where the relationship becomes linear to extract the thermal conductivities.</span>
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<p id="pp">Nagasaka and Nagashima noted that wire insulation has negligible impact on the measurement of the
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thermal conductivity of saline solutions (Nagasaka and Nagashima 1981) and thus can be described by the equation:
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$$ \lambda = \frac{Q}{4\pi\Delta T}\ \ln{(t)}$$ where $Q$ is the power per unit length of the wire,
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$Q = \frac{(I \times V)}{Length}$, and $\Delta T$ is the change in temperature over time $t$.
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  <p id="pp">We found the thermal conductivity of LB and M9 to be similar to that of water,  
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  at 605 $\pm$ 20 $\frac{mW}{Km}\text{ }$ and 570 $\pm$ 30 $\frac{mW}{Km}\text{ }$ respectively.
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  A linear fit of a $T$ vs. $ln(t)$ plot following only the reading from the liquid medium thermocouple
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will yield the conductivity (Fig. 2).</p>
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<h5>References</h5>
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<h5 style="padding-top:15px;"><u>References</u></h5>
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<li>Healy, J.J, de Groot, J.J and Kestin, J, The Theory of the Transient Hot-Wire Method for Measuring Thermal Conductivity,<i>Physica</i> <b>82C</b>: 392-408 (1976)
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<li>Healy, J.J, de Groot, J.J and Kestin, J, The Theory of the Transient Hot-Wire Method for
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<li>Nagasaka Y, Nagashima A. Absolute measurement of the thermal conductivity of electrically conducting liquids by the transient hot-wire method. Journal of Physics E: Scientific Instruments. 1981 Dec;14(12):1435–40.</li>
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Measuring Thermal Conductivity,<i>Physica</i> <b>82C</b>: 392-408 (1976)
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<li>Nagasaka Y, Nagashima A. Absolute measurement of the thermal conductivity of electrically  
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conducting liquids by the transient hot-wire method. Journal of Physics E: Scientific Instruments. 1981 Dec;14(12):1435–40.</li>
 
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<p id="pp">For each team we produced a description of what their project was about (including anything that was particularly notable about the project), the project track, the number of team members, the chassis used (if relevant), any research benchmarks, a list of submitted parts with part numbers, descriptions and links, the medal achieved, and any awards and nominations. Each project was tagged with all relevant keywords, in line with Purdue’s aim of creating a user-friendly, searchable database.</p>
 
<p id="pp">For each team we produced a description of what their project was about (including anything that was particularly notable about the project), the project track, the number of team members, the chassis used (if relevant), any research benchmarks, a list of submitted parts with part numbers, descriptions and links, the medal achieved, and any awards and nominations. Each project was tagged with all relevant keywords, in line with Purdue’s aim of creating a user-friendly, searchable database.</p>
  
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<p id="pp">Purdue conducted a continuous culture of a kill switch that the 2015 Purdue team created but never submitted, and sent us the results of their experiment to give us a broader view of the reliability of kill switches, beyond our current scope of Killer Red and Killer Orange.</p>
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<p id="pp">Purdue conducted a continuous culture of a kill switch that the 2015 Purdue team created but never submitted, and sent us the results of their experiment to give us a broader view of the reliability of kill switches, beyond our current scope of KillerRed and KillerOrange.</p>
  
 
<p id="pp">They followed a continuous culture protocol written by Dan, in which they took the OD of the continuous culture every morning and evening, adding a new flask to return the OD to 0.05 each time to continue the culture. For each day of week 1, and every other day for the weeks following that, they would prepare a glycerol stock of a sample of the culture, then test it in an appropriate way for their kill switch to test if it was still functional, and to what degree it was still functioning. Purdue said once they had substituted out LB broth for yeast YPD media, the protocol was straightforward and easy to follow.</p>
 
<p id="pp">They followed a continuous culture protocol written by Dan, in which they took the OD of the continuous culture every morning and evening, adding a new flask to return the OD to 0.05 each time to continue the culture. For each day of week 1, and every other day for the weeks following that, they would prepare a glycerol stock of a sample of the culture, then test it in an appropriate way for their kill switch to test if it was still functional, and to what degree it was still functioning. Purdue said once they had substituted out LB broth for yeast YPD media, the protocol was straightforward and easy to follow.</p>
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                 <p id="pp">We collaborated with Glasgow iGEM 2016 to test the efficiency of the T7 Promoter we were using to construct the KillerRed, KillerOrange and Lysozyme kill switches. We new that it was leaky and we speculated that it was reducing the efficiency of our project but we needed proof that the leakiness of the promoter could affect our project. In return they gave us a DH5α.Z1 strain in the hopes it would improve the efficiency of our promoter. After subsequent testing we were unable to express our KillerRed and KillerOrange proteins in this strain. This is the report they sent us for the test of the T7 promoter:</p>
 
                 <p id="pp">We collaborated with Glasgow iGEM 2016 to test the efficiency of the T7 Promoter we were using to construct the KillerRed, KillerOrange and Lysozyme kill switches. We new that it was leaky and we speculated that it was reducing the efficiency of our project but we needed proof that the leakiness of the promoter could affect our project. In return they gave us a DH5α.Z1 strain in the hopes it would improve the efficiency of our promoter. After subsequent testing we were unable to express our KillerRed and KillerOrange proteins in this strain. This is the report they sent us for the test of the T7 promoter:</p>
  
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                 <h5>Exeter and Glasgow iGEM 2016 Collaboration: <br \>
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                 KillerRed and KillerOrange Promoter Efficiency Experiment
 
                 KillerRed and KillerOrange Promoter Efficiency Experiment
 
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<p id="pp">Fluorescence scan image from the Typhoon with labels for which samples are in each well.</p>
 
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                 <p id="pp">These data indicate that there is no difference in fluorescence between either KillerRed or KillerOrange and the cells only control either with or without induction with IPTG. There could be several reasons for this, including the light was not intense enough to excite the fluorescent proteins, however no fluorescence from this type of the test with a laser for excitation would be unlikely.  It is also possible that no protein is being produced, which could be due to insufficient IPTG. However, the RFP in J04450 under the control of the lac-repressible promoter R0010 clearly shows that in the DH5α.Z1 strain, there is less fluorescence without IPTG, than with IPTG. This is not a perfect control for the concentration of IPTG used unless KillerRed and KillerOrange also have the R0010 promoter. Interestingly, in the DH5α strain, there is no significant difference between RFP fluorescence with or without IPTG – this is due to DH5α not having a functional copy of LacI, the lac repressor, therefore lac-repressible promoters are not “OFF”, so cannot be switched “ON” by IPTG induction. </p>
 
                 <p id="pp">These data indicate that there is no difference in fluorescence between either KillerRed or KillerOrange and the cells only control either with or without induction with IPTG. There could be several reasons for this, including the light was not intense enough to excite the fluorescent proteins, however no fluorescence from this type of the test with a laser for excitation would be unlikely.  It is also possible that no protein is being produced, which could be due to insufficient IPTG. However, the RFP in J04450 under the control of the lac-repressible promoter R0010 clearly shows that in the DH5α.Z1 strain, there is less fluorescence without IPTG, than with IPTG. This is not a perfect control for the concentration of IPTG used unless KillerRed and KillerOrange also have the R0010 promoter. Interestingly, in the DH5α strain, there is no significant difference between RFP fluorescence with or without IPTG – this is due to DH5α not having a functional copy of LacI, the lac repressor, therefore lac-repressible promoters are not “OFF”, so cannot be switched “ON” by IPTG induction. </p>
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                 <p id="pp">Both plasmids had the BioBrick prefix, and the correct sequence for both KillerRed and KillerOrange open reading frame, according to the papers cited on the Exeter 2016 iGEM wiki. The sequence between the prefix and the ATG start codon, we checked against lac-repressible promoters in the iGEM registry. We found a match to R0184, which is a T7 lac-repressible promoter. T7 promoters require T7 polymerase to be transcribed, as they are not recognised by E. coli polymerases. This results confirms the result of the fluorescence measurements. No KillerRed or KillerOrange protein was observed by fluorescence, as neither gene was being transcribed by either DH5α or DH5α.Z1 as neither strain produces the required T7 polymerase. A protein overexpression E. coli strain such as BL21<DE3> which has the T7 polymerase gene inserted into its genome is designed to use T7 promoters would have been able to express these KillerRed and KillerOrange constructs.</p>
 
                 <p id="pp">Both plasmids had the BioBrick prefix, and the correct sequence for both KillerRed and KillerOrange open reading frame, according to the papers cited on the Exeter 2016 iGEM wiki. The sequence between the prefix and the ATG start codon, we checked against lac-repressible promoters in the iGEM registry. We found a match to R0184, which is a T7 lac-repressible promoter. T7 promoters require T7 polymerase to be transcribed, as they are not recognised by E. coli polymerases. This results confirms the result of the fluorescence measurements. No KillerRed or KillerOrange protein was observed by fluorescence, as neither gene was being transcribed by either DH5α or DH5α.Z1 as neither strain produces the required T7 polymerase. A protein overexpression E. coli strain such as BL21<DE3> which has the T7 polymerase gene inserted into its genome is designed to use T7 promoters would have been able to express these KillerRed and KillerOrange constructs.</p>
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                 <p id="pp">Glasgow iGEM did fantastic work for us, providing us with detailed analysis of the T7 promoter and suggestions for improving the efficiency of our project. Whilst their data on the DH5alpha Z1 strain is accurate and in accordance with subsequent research and advice, we have since noted there is expression of KillerRed and KillerOrange in DH5alpha in lab tests.  </p>
 
                 <p id="pp">Glasgow iGEM did fantastic work for us, providing us with detailed analysis of the T7 promoter and suggestions for improving the efficiency of our project. Whilst their data on the DH5alpha Z1 strain is accurate and in accordance with subsequent research and advice, we have since noted there is expression of KillerRed and KillerOrange in DH5alpha in lab tests.  </p>
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<p id="pp">We are proud and grateful in saying that we have helped and been helped by various other teams in this years iGEM competition. Utilising other teams equipment and different ways of thinking towards solving problems has provided us all with some great data and innovative solutions to some problems we have encountered. We are confident that what we have achieved will benefit the iGEM competition as a whole. </p>
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<p id="pp">Good inter-team communication is a vital factor in all disciplines and at the core of the iGEM spirit. We hope to have contributed to this in several ways. </p>
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Theory: Edinburgh
 
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<h6>Optimising methods of data mutation detection in BabbleBlocks</h6>
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<h6>Optimising methods of data mutation detection in BabbleBlocks</h6>
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Edinburgh’s 2016 under-graduate team is utilising the natural information storage capability of DNA to store digital information. Currently they have outlined a method for using a base 4 system to store words in biological material called “BabbleBlocks”. During the Westminster UK iGEM meetup Edinburgh talked about the issue of mutated DNA going relatively undetected. Their method at the time involved adding up the value of each base and storing this as a “Checksum” at the end of the BabbleBlock. Edinburgh’s team highlighted in their presentation that they knew this would lead to a large amount of false positives as many different combinations of information could add to the same Checksum. My research looks in to the method of the Checksum and attempts to create a new method that has a smaller chance of false positives.
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Storing information on DNA offers many advantages over current methods, however mutations  
 
Storing information on DNA offers many advantages over current methods, however mutations  
 
need to be carefully monitored to ensure incorrect data is not read as a false positive.  
 
need to be carefully monitored to ensure incorrect data is not read as a false positive.  
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If continued further, research should also be done in to the reconstruction of data after it has been lost.
 
If continued further, research should also be done in to the reconstruction of data after it has been lost.
 
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Stanford Brown
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  We initiated contact with Stanford-Brown this year with the intention of interviewing one of their supervisors, Prof. Lynn Rothschild. We were interested in hearing     
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  about her opinions as well as experiences with diversity and equality in science. Unfortunately we were never able to find a mutually suitable date to carry out the 
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  interview. However we were able to organise a collaboration with her team.
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  The Stanford-Brown team have helped our podcast and YouTube series by making their own edition of Desert Island… Science? Which is now available on our 
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  YouTube and SoundCloud channels.
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<source src="https://static.igem.org/mediawiki/2016/7/7f/T--Exeter--Collab_stanfordaudioogg.ogg" type="audio/ogg">
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<source src="https://static.igem.org/mediawiki/2016/a/a7/T--Exeter--Log_31.5.16vlogmp3.mp3" type="audio/mpeg">
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<p id="pp">
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  We are very thankful to Stanford-Brown for their contribution, as it has helped our work get further recognition on an international scale.
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<p id="pp">We are also proud and grateful in saying that we have helped and been helped by various other teams in this years iGEM competition. Utilising other teams equipment and different ways of thinking towards solving problems has provided us all with some great data and innovative solutions to some problems we have encountered. We are confident that what we have achieved will benefit the iGEM competition as a whole. </p>
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<p id="pp">Good inter-team communication is a vital factor in all disciplines and at the core of the iGEM spirit. We hope to have contributed to this in several ways. </p>
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Latest revision as of 02:43, 20 October 2016