<commit_before>---
layout: page
title: "The Project"
sub-title: "The Project"
description: ""
header-img: "img/home2-bg.jpg"
sections:
- Background
- The project idea
- Goals

---

<section id="Background">

<h1>Background</h1>

<p> Cellular processes are highly dependent on metabolite concentrations and even small perturbations can lead to cell differentiation, apoptosis or cancer. <sup>1</sup> A biosensor system that allows determination of metabolite concentrations <i>in vivo</i> is therefore a valuable tool.  </p>

<p> An aptamer is an nucleic acid or a peptide molecule that binds to a target with affinities and specificities that rival those of antibodies. Optical aptamers are capable of inducing fluorescence in a target molecule upon binding. An example of this is the RNA aptamer called Spinach (discovered by Paige, et. al. in 2011, enhanced by Strack, et. al. in 2013).<sup>2,3</sup> This aptamer binds the molecule 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) (see animation) and causes it to fluoresce at a wavelength of 495 nm. It has an fluorescent enhancement that rivals that of green fluorescent protein and almost no photobleaching.</p>

<p><img src="{{ site.baseurl }}/img/theproject/gif1.gif" width="60%" class="img-responsive img-center">
<div class="row">
	<div class="col-md-10 col-md-offset-1 figure-text">Figure 1: Visualization of the binding of DFHBI to Spinach</div>
</div>

<p>In 2012 Paige et. al. found that fusing the Spinach aptamer with an s-adenosyl methionine (SAM) aptamer (as seen in Figure 2) made detection of SAM possible. They furthermore showed that other small-molecule aptamers work in the system showing its great flexibility.</p>

<img src="{{ site.baseurl }}/img/theproject/theoriginalconstruct1.png" class="img-responsive img-center">
<div class="row">
	<div class="col-md-10 col-md-offset-1 figure-text">Figure 2: The original SAM sensor as described by Paige et. al in 2012. The sensor RNA consists of the optical aptamer Spinach (black), a linker region (orange) and a detection aptamer (blue). When the aptamer binds its target, stabilization of the linker region is promoted, enabling Spinach to bind DFHBI and activate fluorescence. Picture retrieved from [4]. </div>
</div>


</section>


<section id="Theprojectidea">
<div class="border"></div>
<h1>The Project Idea</h1>

<p> Our concentration dependent biosensor system is based on the aptamer system that Paige et. al.<sup>4</sup> They combined a small molecule dependent aptamer with Spinach thereby creating a small molecule sensor. However, the sensor has a limited detection range. The ultimate goal of the project was to develop a small molecule-sensor with a broadened dynamic range which works <i>in vivo</i>. We wanted to do this by making RNA sensors with different affinities for the same target molecule. These can be combined to improve the dynamic range of the sensor. We furthermore wanted to include a internal control for the level of transcription, as this can vary from cell to cell and result in an inaccurate readout. We therefore need an orthogonal optical aptamer, which would fluoresce independently of the target molecule concentration.</p>

<p>We aim to achieve this by designing different small molecule sensors with different stabilities towards the target molecule and furthermore investigate the orthogonality.</p>    

<img src="{{ site.baseurl }}/img/theproject/Projectidea.png" class="img-responsive img-center">
<div class="row">
	<div class="col-md-10 col-md-offset-1 figure-text">Figure 3: 
	</div>
</div>

</section>



<section id="Goals">
<div class="border"></div>
<h1>Goals</h1>

The overall goal of the project was to create a concentration dependent biosensor. The biosensor should consist of multiple small molecule dependent aptamers with different affinities for the target creating a broader dynamic range. To ensure a reliable readout in a cellular setting the sensor must contain an internal control for the transcription level.
In order to achieve this the following subgoals had to be reached:
1. Design and test aptamer sensors with different concentration dependency
2. Evaluate fluorophore-aptamer pairs to create an orthogonal internal control for the transcription level
3. Setup an evaluation platform for testing the biosensor <i>in vivo</i>   

We devised the following workflow:

<img src="{{ site.baseurl }}/img/theproject/Flowchart2.png" class="img-responsive img-center">


	
</ul>

</section>

<div class="border"></div>

<p><h1>References</h1></p>

<ol type="1">
<li> <NAME>; <NAME>; <NAME>; <NAME>; <NAME>; <NAME>.  <i> Molecular Biology of The Cell 5th Edition.</i>, Garland Science, Abingdon. </li>

<li> <NAME>., <NAME>., and <NAME>. (2011) RNA mimics of green fluorescent protein. <i>Science</i> <b>333</b>, 642.</li>

<li> <NAME>., <NAME>., and <NAME>. (2013) A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. <i>Nat. Methods</i> <b>10</b>, 1219−1224</li>

<li> <NAME>., <NAME>., <NAME>, <NAME>. (2012) Fluorescence Imaging of Cellular Metabolites with RNA. <i>Science<i>, <b>335<b> 1194 </li>

</ol>








<commit_msg>project upd<commit_after>---
layout: page
title: "The Project"
sub-title: "The Project"
description: ""
header-img: "img/home2-bg.jpg"
sections:
- Background
- The project idea
- Goals

---

<section id="Background">

<h1>Background</h1>

<p> Cellular processes are highly dependent on metabolite concentrations and even small perturbations can lead to cell differentiation, apoptosis or cancer. <sup>1</sup> A biosensor system that allows determination of metabolite concentrations <i>in vivo</i> is therefore a valuable tool.  </p>

<p> An aptamer is an nucleic acid or a peptide molecule that binds to a target with affinities and specificities that rival those of antibodies. Optical aptamers are capable of inducing fluorescence in a target molecule upon binding. An example of this is the RNA aptamer called Spinach (discovered by Paige, et. al. in 2011, enhanced by Strack, et. al. in 2013).<sup>2,3</sup> This aptamer binds the molecule 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) (see animation) and causes it to fluoresce at a wavelength of 495 nm. It has an fluorescent enhancement that rivals that of green fluorescent protein and has almost no photobleaching.</p>

<p><img src="{{ site.baseurl }}/img/theproject/gif1.gif" width="60%" class="img-responsive img-center">
<div class="row">
	<div class="col-md-10 col-md-offset-1 figure-text">Figure 1: Visualization of the binding of DFHBI to Spinach</div>
</div>

<p>In 2012 Paige et. al. found that fusing the Spinach aptamer with an s-adenosyl methionine (SAM) aptamer (as seen in Figure 2) made detection of SAM possible. They furthermore showed that other small-molecule aptamers work in the system showing its great flexibility.</p>

<img src="{{ site.baseurl }}/img/theproject/theoriginalconstruct1.png" class="img-responsive img-center">
<div class="row">
	<div class="col-md-10 col-md-offset-1 figure-text">Figure 2: The original SAM sensor as described by Paige et. al in 2012. The sensor RNA consists of the optical aptamer Spinach (black), a linker region (orange) and a detection aptamer (blue). When the aptamer binds its target, stabilization of the linker region is promoted, enabling Spinach to bind DFHBI and activate fluorescence. Picture retrieved from [4]. </div>
</div>


</section>


<section id="Theprojectidea">
<div class="border"></div>
<h1>The Project Idea</h1>

<p> Our concentration dependent biosensor system is based on the aptamer system that Paige et. al.<sup>4</sup> They combined a small molecule dependent aptamer with Spinach thereby creating a small molecule sensor. However, the sensor has a limited detection range. The ultimate goal of the project was to develop a small molecule-sensor with a broadened dynamic range which works <i>in vivo</i>. We wanted to do this by making RNA sensors with different affinities for the same target molecule. These can be combined to improve the dynamic range of the sensor. We furthermore wanted to include a internal control for the level of transcription, as this can vary from cell to cell and result in an inaccurate readout. We therefore need an orthogonal optical aptamer, which would fluoresce independently of the target molecule concentration.</p>

<p>We aim to achieve this by designing different small molecule sensors with different stabilities towards the target molecule and furthermore investigate the orthogonality.</p>    

<img src="{{ site.baseurl }}/img/theproject/Projectidea.png" class="img-responsive img-center">
<div class="row">
	<div class="col-md-10 col-md-offset-1 figure-text">Figure 3: 
	</div>
</div>

</section>



<section id="Goals">
<div class="border"></div>
<h1>Goals</h1>

The overall goal of the project was to create a concentration dependent biosensor. The biosensor should consist of multiple small molecule dependent aptamers with different affinities for the target creating a broader dynamic range. To ensure a reliable readout in a cellular setting the sensor must contain an internal control for the transcription level.
In order to achieve this the following subgoals had to be reached:
1. Design and test aptamer sensors with different concentration dependency
2. Evaluate fluorophore-aptamer pairs to create an orthogonal internal control for the transcription level
3. Setup an evaluation platform for testing the biosensor <i>in vivo</i>   

We devised the following workflow:

<img src="{{ site.baseurl }}/img/theproject/Flowchart2.png" class="img-responsive img-center">


	
</ul>

</section>

<div class="border"></div>

<p><h1>References</h1></p>

<ol type="1">
<li> <NAME>; <NAME>; <NAME>; <NAME>; <NAME>; <NAME>.  <i> Molecular Biology of The Cell 5th Edition.</i>, Garland Science, Abingdon. </li>

<li> <NAME>., <NAME>., and <NAME>. (2011) RNA mimics of green fluorescent protein. <i>Science</i> <b>333</b>, 642.</li>

<li> <NAME>., <NAME>., and <NAME>. (2013) A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. <i>Nat. Methods</i> <b>10</b>, 1219−1224</li>

<li> <NAME>., <NAME>., <NAME>. (2012) Fluorescence Imaging of Cellular Metabolites with RNA. <i>Science<i>, <b>335<b> 1194 </li>

</ol>









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