Difference between revisions of "Team:Rice/DrylabAnalysis"

 
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         <h1></h1>
 
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        <h2>
−
           
+
            Software Conclusions
−
                <p><br />
+
        </h2>
−
                    Existing RNA thermometers operate around 37°C and do not exhibit significant conformational changes between 25°C and 30°C. As most plants will die
+
        <p>
−
                    at 37°C, it was necessary to design thermometers that experienced conformational change between 25°C. We chose to design thermometers by finding optimal
+
            As shown below, the experimental tests of the software-designed RNA thermometers demonstrated that
−
                    candidates using a genetic algorithm, as the complexity of RNA folding made it difficult to rationally design thermometers.
+
            some could indeed outperform iGEM kit thermometers created through more conventional methods. From this,
−
                </p>
+
            we can conclude that genetic algorithms are viable methods to design custom RNA thermometers when paired
 +
            with software such as NuPack which can predict basic properties of RNA thermometers.
 +
        </p>
 +
        <p>
 +
            In the future, the fitness functions could be better optimized to predict the performance of RNA thermometers.
 +
            Also, there is a large body on evolutionary algorithms that the software team was not able to explore, some of
 +
            which may allow for better performance.
 +
        </p>
 +
    </div>
  
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<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2019/e/e0/T--Rice--arabidopsisgrowingtemp.svg"/>
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            <figure>
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                <img class="img-responsive" style="width: 70%;"
−
  <img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2019/d/dd/T--Rice--putidagrowingtemp.svg"/>    
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                    src="https://static.igem.org/mediawiki/2019/1/11/T--Rice--3025v2.png" />
−
  </div>  
+
                <figcaption>
−
<br />
+
                    This figure shows the performance of all the tested thermometers that performed better than
−
<br />
+
                    our positive controls at the temperature range. As we measured the thermometer performance based on mCherry expression, the
−
<br />
+
                    y-axis shows the ratio of mCherry expression at the temperature bounds.(30°C/25°C). "NC" stands for
−
<br />
+
                    No Chill, which is the collective name for the RNA thermometer parts our team released. "V"
−
</div>
+
                    thermometers
 +
                    were designed but did not perform well enough to be included as parts. "K" thermometers were provided
 +
                    with the iGEM kit.
 +
                </figcaption>
 +
            </figure>
  
−
<div class="row" style = "background-color:#26849c!important;">
+
            <figure>
−
</div><h2 style = "background-color:#1f9ec1!important;"> RNA Thermometer Design </h2> </br>
+
                <img class="img-responsive" style="width: 70%;"
 +
                    src="https://static.igem.org/mediawiki/2019/6/68/T--Rice--new30-26.png" />
 +
                <figcaption>
 +
                    This figure shows the performance of certain the tested thermometers that performed better than
 +
                    our positive controls at the temperature range. As we measured the thermometer performance based on mCherry expression, the
 +
                    y-axis shows the ratio of mCherry expression at the temperature bounds.(30°C/25°C). "NC" stands for
 +
                    No Chill, which is the collective name for the RNA thermometer parts our team released. "V"
 +
                    thermometers
 +
                    were designed but did not perform well enough to be included as parts. "K" thermometers were provided
 +
                    with the iGEM kit.
 +
                </figcaption>
 +
            </figure>
 +
 
         </div>
 
         </div>
 
</div>
 
</div>
−
<div class="row" style = "background-color:#fff!important;">  
+
<div class="row" , style="background-color: #d28ec5!important;" align="center">
−
<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2019/8/8c/T--Rice--thermometerlocky.svg"/>
+
        <div class="row" , style="background-color:#d2e8c5!important;">
−
<p>In essence, RNA thermometers are a form of temperature dependent translational regulation. At low temperatures, there is a higher probability of more base pairs forming what is known as a "stem-loop structure". At higher temperatures, the thermometers "melt", meaning there is a decreased likelihood of base pairs forming. </p>
+
            <figure>
 +
                <img class="img-responsive" style="width: 70%;"
 +
                    src="https://static.igem.org/mediawiki/2019/0/0b/T--Rice--3730v2.png" />
 +
                <figcaption>
 +
                        This figure shows the performance of all the tested thermometers that performed better than
 +
                        our positive controls at the temperature range. As we measured the thermometer performance based on mCherry expression, the
 +
                        y-axis shows the ratio of mCherry expression at the temperature bounds.(37°C/30°C). "NC" stands for
 +
                        No Chill, which is the collective name for the RNA thermometer parts our team released. "V"
 +
                        thermometers
 +
                        were designed but did not perform well enough to be included as parts. "K" thermometers were provided
 +
                        with the iGEM kit.
 +
                </figcaption>
 +
            </figure>
 +
        </div>
 
</div>
 
</div>
−
<div class="column" style="background-color:#fff!important;">
+
<div class="row" , style="background-color: #d28ec5!important;" align="center">
−
<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2019/0/01/T--Rice--foldedthermometerdesign.svg"/>
+
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−
</div>
+
             <figure>
−
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+
                <img class="img-responsive" style="width: 70%;"
−
<p>The stem-loop structure of the thermometer was created by taking the complement of the ribosome binding site(RBS) and surrounding the nucleotides and introducing mutations into the sequence. The resulting altered sequence is known as the variable region. Mutating this region serves a two prong purpose. First off, it ensures that the RBS will be locked into the structure of the thermometer, rendering it inaccessible to the ribosome at low temperatures. Secondly, it allows us to optimize for the targeted melting temperature. </p>
+
                    src="https://static.igem.org/mediawiki/2019/6/6e/T--Rice--3725v2.png" />
−
             </div>
+
                 <figcaption>
−
            </div>
+
                         This figure shows the performance of all the tested thermometers that performed better than
−
           
+
                         our positive controls at the temperature range. As we measured the thermometer performance based on mCherry expression, the
−
<div class="row" style = "background-color:#fff!important; padding-top:5vh;">
+
                         y-axis shows the ratio of mCherry expression at the temperature bounds.(37°C/25°C). "NC" stands for
−
<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2019/4/49/T--Rice--thermometerdesignn.svg"/>
+
                         No Chill, which is the collective name for the RNA thermometer parts our team released. "V"
−
</div> <div class="row" style = "background-color:#9bc4cf!important; padding-top:5vh;">
+
                         thermometers
−
            <h2>Percentage base pair optimization algorithm</h2>
+
                         were designed but did not perform well enough to be included as parts. "K" thermometers were provided
−
            <ol>
+
                         with the iGEM kit.
−
                 <li>
+
                 </figcaption>
−
                    <p>
+
             </figure>
−
                         For each base of the complement of the context containing the RBS, create three other
+
        </div>
−
                         permutations which have that base mutated. All of these permutations combined form the initial
+
−
                        population. The baseline is defined as the full sequence containing the context before the
+
−
                        variable region, the variable region which is the complement of the RBS-containing context, and
+
−
                         the RBS-containing context.
+
−
                    </p>
+
−
                </li>
+
−
                <li>
+
−
                    <p>
+
−
                        Calculate the base pairing probabilities at the two given temperatures for each test sequence
+
−
                        using the NuPack command <code>pairs -T TEMP -pseudo -material rna sequencename.</code>
+
−
                    </p>
+
−
                </li>
+
−
                <li>
+
−
                    <p>
+
−
                        The output of the command consists in part of a list of every base and the base(s) which it has
+
−
                        a > 0.001 probability of base pairing with by position number. Find the base pairings where one
+
−
                         of the bases is in the RBS-containing region and add up the probabilities corresponding to that
+
−
                        base pairing. The nature of this NuPack command should prevent duplicates. Then, subtract the
+
−
                         probability that these bases are unpaired.
+
−
                    </p>
+
−
                </li>
+
−
                <li>
+
−
                    <p>
+
−
                        Subtract the number of base pairings at 30°C from the number of base pairings at 25°C. We want
+
−
                         to maximize the number of RBS-base pairings that disappear as the temperature increases from
+
−
                         25°C to 30°C (for reference, the command I use for the minimum free energy calculation is
+
−
                        <code>mfe -T TEMP -pseudo -material rna sequencename</code>)
+
−
                    </p>
+
−
                </li>
+
−
                <li>
+
−
                    <p>
+
−
                        Using the library DEAP, select 50 individuals through the tournament selection method.
+
−
                    </p>
+
−
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+
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+
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+
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{{Rice/footer}}

Latest revision as of 03:06, 14 December 2019

Software Conclusions

As shown below, the experimental tests of the software-designed RNA thermometers demonstrated that some could indeed outperform iGEM kit thermometers created through more conventional methods. From this, we can conclude that genetic algorithms are viable methods to design custom RNA thermometers when paired with software such as NuPack which can predict basic properties of RNA thermometers.

In the future, the fitness functions could be better optimized to predict the performance of RNA thermometers. Also, there is a large body on evolutionary algorithms that the software team was not able to explore, some of which may allow for better performance.

This figure shows the performance of all the tested thermometers that performed better than our positive controls at the temperature range. As we measured the thermometer performance based on mCherry expression, the y-axis shows the ratio of mCherry expression at the temperature bounds.(30°C/25°C). "NC" stands for No Chill, which is the collective name for the RNA thermometer parts our team released. "V" thermometers were designed but did not perform well enough to be included as parts. "K" thermometers were provided with the iGEM kit.
This figure shows the performance of certain the tested thermometers that performed better than our positive controls at the temperature range. As we measured the thermometer performance based on mCherry expression, the y-axis shows the ratio of mCherry expression at the temperature bounds.(30°C/25°C). "NC" stands for No Chill, which is the collective name for the RNA thermometer parts our team released. "V" thermometers were designed but did not perform well enough to be included as parts. "K" thermometers were provided with the iGEM kit.
This figure shows the performance of all the tested thermometers that performed better than our positive controls at the temperature range. As we measured the thermometer performance based on mCherry expression, the y-axis shows the ratio of mCherry expression at the temperature bounds.(37°C/30°C). "NC" stands for No Chill, which is the collective name for the RNA thermometer parts our team released. "V" thermometers were designed but did not perform well enough to be included as parts. "K" thermometers were provided with the iGEM kit.
This figure shows the performance of all the tested thermometers that performed better than our positive controls at the temperature range. As we measured the thermometer performance based on mCherry expression, the y-axis shows the ratio of mCherry expression at the temperature bounds.(37°C/25°C). "NC" stands for No Chill, which is the collective name for the RNA thermometer parts our team released. "V" thermometers were designed but did not perform well enough to be included as parts. "K" thermometers were provided with the iGEM kit.