Difference between revisions of "Team:BostonU/Results"

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  <p class="wide-heading-type mainwrap align-center">RESULTS</p>
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</div></div>
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<div id="panel1" class="link-slideup">
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  <p class="inline-heading-type mainwrap">Characterizing Our Cell Free System</p>
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  <p class="body-type mainwrap">The first step in our project was to characterize the activity of the cell-free transcription-translation system we made in house. This characterization required measuring the maximum protein expression levels that can be accomplished in the cell free system. We first designed a mathematical model to estimate the concentration of DNA at which fluorescence would saturate in the system. We found that the expression capacity of the system is best modeled as a bell-shaped dose response curve, which we describe in greater detail on our modeling page. We experimentally characterized the cell-free system by measuring the expression of varying concentrations of a plasmid coding for a constitutively active deGFP gene. We found that maximal expression in the cell-free system is achieved around 20 nM concentrations of DNA.</p>
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  <p class="body-type mainwrap">&nbsp;</p>
  
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  <div class="figwrap mainwrap body-type">
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  <img src="https://static.igem.org/mediawiki/2017/6/60/T--BostonU--MaxDNAData.png"></img>
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  <p class="body-type"><strong>Figure 1.</strong>This figure shows fluorescence from constitutive deGFP plasmids at 10 nM, 20 nM, 30 nM, and 40 nM concentrations as well as a reaction containing no DNA. </p>
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  </div>
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  <p class="body-type mainwrap">&nbsp;</p>
  
<div class="column full_size" >
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  <p class="inline-heading-type mainwrap">Characterizing Toehold Switch Activity in Cell Free</p>
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  <p class="body-type mainwrap"> The next step in validating our project was to characterize toehold switch functionality in the cell-free system. The best expression was achieved when activating the toehold switch with RNA trigger at 10,000X concentrations relative to the toehold. In this experiment, fluorescence from reactions with plasmid toeholds and RNA trigger was compared to fluorescence from reactions containing plasmid toehold switches alone, no DNA at all, and a plasmid containing a constitutively active deGFP. Measurable fluorescence is achieved from two different toehold switch/trigger pairs, but the expression is lower than that seen from the constitutively active deGFP. Greater amounts of input RNA may allow for better fluorescence, but the methods we employed for transcribing the trigger DNA to RNA were not able to produce higher concentrations of RNA.</p>
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  <p class="body-type mainwrap">&nbsp;</p>
  
<h1>Results</h1>
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  <div class="figwrap mainwrap body-type">
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  <img src="https://static.igem.org/mediawiki/2017/2/2f/T--BostonU--THDataBoth.png"></img>
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  <p class="body-type"><strong>Figure 2.</strong> This figure shows fluorescence from a cell free reaction in which 10 nM toehold plasmids express deGFP in response to RNA triggers at 10,000X concentrations. This is compared to reactions containing no DNA, only toehold plasmid DNA, and a plasmid with constitutively active deGFP. </p>
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  </div>
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  <p class="body-type mainwrap">&nbsp;</p>
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  <p class="body-type mainwrap">Through these results we determined that toehold switches produce detectable levels of protein expression when activated by trigger RNA at 10,000X concentrations. Future experiments would aim to improve expression.
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  <p class="body-type mainwrap">&nbsp;</p>
  
<p>Here you can describe the results of your project and your future plans. </p>
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  <p class="inline-heading-type mainwrap"> Characterizing Recombinase Activity in Cell Free </p>
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  <p class="body-type mainwrap"> Once toehold switches were functional in the cell-free system, we shifted to cloning recombinase genes that could function in our cell free system. We first characterized the performance of commercially available Cre recombinase (NEB) in cell-free. Using a terminator excision reporter plasmid (shown below), the Cre recombinase should excise the premature terminator and allow for downstream deGFP expression. When fluorescence from this reporter plasmid with added Cre recombinase is compared to fluorescence from a reaction with no DNA and a reaction with constitutively active deGFP, only background levels of fluorescence are seen from the reporter. In order to explore this negative result further, we set up a reaction with Cre recombinase and the constitutively active deGFP plasmid. The goal of this experiment is to observe how the Cre recombinase itself affects the transcriptional and translational activity of the cell-free system.  In this experiment, we saw that the fluorescence level achieved by the constitutively active deGFP was only 25% of the fluorescence levels achieved without recombinases in the solution. </p>
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  <p class="body-type mainwrap">&nbsp;</p>
  
<h5>What should this page contain?</h5>
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  <div class="figwrap mainwrap body-type">
<ul>
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  <img src="https://static.igem.org/mediawiki/2017/4/48/T--BostonU--CreData.png"></img>
<li> Clearly and objectively describe the results of your work.</li>
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  <p class="body-type"><strong>Figure 3.</strong>This figure shows fluorescence from a cell free reaction in which 1 unit of Cre recombinase was added to 10 nM reporter plasmids in order to drive deGFP expression. This is compared to reactions containing no DNA, only reporter plasmid DNA, a plasmid with constitutively active deGFP, and a reaction with 1 unit of Cre added to the constitutive deGFP plasmid. </p>
<li> Future plans for the project. </li>
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  </div>
<li> Considerations for replicating the experiments. </li>
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</ul>
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<h5>You should also describe what your results mean: </h5>
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  <p class="body-type mainwrap">&nbsp;</p>
  
<ul>
 
<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
 
<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
 
<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
 
</ul>
 
  
</div>
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  <p class="body-type mainwrap">Our next step was to determine if similar results occurred when testing with plasmid contained recombinases. We tested a plasmid-bound BxbI recombinase and a reporter plasmid with an inverted promoter (shown below). The BxbI recombinase should put the promoter in the proper orientation, allowing for deGFP expression. Fluorescence from a reaction containing recombinase and reporter again shows only background levels of fluorescence as compared to a reaction with no DNA. A reaction containing the recombinase and the constitutively active deGFP showed expression at about 66% as compared to a reaction containing just a constitutively active deGFP plasmid. In this case, we believe that the decrease may be caused by transcription and translation machinery is being diverted to produce the recombinase. Future experiments will be aimed at testing additional types of recombinases in cell free and understanding in more detail the mechanisms leading to the decreased deGFP expression.  </p>
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  <p class="body-type mainwrap">&nbsp;</p>
  
<div class="clear"></div>
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    <div class="figwrap mainwrap body-type">
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  <img src="https://static.igem.org/mediawiki/2017/5/5e/T--BostonU--BxbIData.png"></img>
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  <p class="body-type"><strong>Figure 4.</strong> This figure shows fluorescence from a cell free reaction in which a constitutive BxbI recombinase plasmid was added to 10 nM reporter plasmids in order to drive deGFP expression. This is compared to reactions containing no DNA, only reporter plasmid DNA, a plasmid with constitutively active deGFP, and a reaction with the constitutive BxbI recombinase plasmid added to the constitutive deGFP plasmid. </p>
 +
  </div>
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  <p class="body-type mainwrap">&nbsp;</p>
  
<div class="column half_size" >
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  <p class="body-type mainwrap">These experiments reveal that at this point, the recombinases we used (Cre and BxbI) do not exhibit detectable rates of recombination in our cell free system. Future experiments will focus on determining failure points in the experiments presented here, and subsequently characterizing functional recombinases in our cell free.
 
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  <p class="body-type mainwrap">&nbsp;</p>
 
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<h5> Project Achievements </h5>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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<ul>
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<li>A list of linked bullet points of the successful results during your project</li>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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</ul>
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<script> /* THIS MAKES ANY WRAPPED ELEMENT ON THE PAGE FADE IN */
 
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<h5>Inspiration</h5>
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    $('.mainwrap').fadeIn(1000);
<p>See how other teams presented their results.</p>
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});
<ul>
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</script>
<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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</body>
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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</ul>
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Latest revision as of 17:21, 1 November 2017

RESULTS