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project.html
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<!doctype html>
<html lang="en">
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<meta charset="UTF-8" />
<link rel="stylesheet" href="css/gumby.css" />
<title>uOttawa iGEM 2013 - Project</title>
<script src="js/jquery-1.9.1.min.js"></script>
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<div id="menubar">
<a href="http://www.igem.org"><img src="images/igem-logo.png" /></a>
<a href="index.html">Home</a>
<a href="project.html">Wet Lab</a>
<a href="software.html">Software</a>
<a href="humanpractices.html">Human Practices</a>
<a href="team.html">Team</a>
<a href="parts.html">Parts</a>
<a href="notebook.html">Notebook</a>
<a href="safety.html">Safety</a>
<a href="attributions.html">Attributions</a>
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<a class="paneLink selected" href=".intro">Introduction</a>
<a class="paneLink" href=".indepth">In-Depth</a>
<a class="paneLink" href=".data">Data</a>
<a class="paneLink" href=".modelling">Modelling</a>
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<h1>Background</h1>
<h2>The Type-I Incoherent Feedforward Loop and Fold-Change Detection</h2>
<p>The type-I incoherent feedforward loop (I1-FFL) is a gene network in which protein X activates a gene Z while simultaneously activating the production of a repressor of gene Z, labeled Y.</p>
<div class="figure">
<img src="images/figures/ffl-simple.png" alt="Type-1 incoherent feed-forward loop" />
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<p>For this year’s project, we are aiming to construct an I1-FFL that can detect fold-changes in the levels of protein X. In other words, the expression of Z would be reliant on the relative change in X as opposed to absolute values of X. For example, if X changes from an arbitrary concentration of 1 to an arbitrary concentration of 5, the level of expression of gene Z would be exactly the same as that resulting from a change from a concentration of 5 to a concentration of 25 of X (a fold-change by a factor of 5 in both cases).</p>
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<h2>How Fold-Change Detection is Achieved</h2>
<p>This fold-change detection is enabled through the repressor Y. Take the case when activator X, repressor Y, and protein Z each have an arbitrary concentration of 1 in the cell (1:1:1 ratio). If the concentration of X is doubled, the ratio between X and Y now becomes 2:1. X immediately activates Z, but since the repressor Y takes time to fold, it lags behind, and the concentration of Z spikes to 2 due to the activation. When Y folds into its active form and carries out its repressive activity, the concentration of Z returns to its initial concentration of 1. This is because at this point, X and Y return to a 1:1 ratio in the cell. In order for the concentration of Z to reach 2 once again, the X:Y ratio must first return to 2:1 - thus, X has to reach a concentration of 4 before the same output of Z is produced. In this way, fold-change detection is achieved.</p>
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<img src="images/figures/ffl-indepth.png" alt="Type-1 incoherent feed-forward loop" />
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<h1>Engineering our System</h1>
<h2>The Design of our Gene Network and the Interactions Between Components</h2>
<p>In our proposed design, rtTA will act as our activator (X), Lacl as our repressor (Y), and GFP as our reporter protein (Z). Each part is detectable via a different fluorescent protein. A visual representation of our pathway is shown in Figure 3.</p>
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<img src="images/figures/networkdesign.png" alt="Type-1 incoherent feed-forward loop" />
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<p>This network design allows for calibration of the network via tunable levels of β-estradiol, allowing for accurate control of the amount of rtTA(X) in the cell. This will allow us to ensure that fold-change detection works as theorized in our network.</p>
<p>Furthermore, activation by rtTA is mediated by varying levels of anhydrotetracycline (ATc), and LacI repression is mediated by varying levels of Isopropyl β-D-1-thiogalactopyranoside (IPTG). These methods of mediation allow for high tunability of the system, and we should be able to find a concentration range of ATc and IPTG at which the system will work as desired.</p>
<p>The system will then be characterized extensively. The fluorescent markers tagged to each component of the network will allow for simple temporal analysis using flow cytometry. This will allow for a high-throughput analysis of the activity of our network.</p>
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<h1>Potential Applications</h1>
<h2>A Fold-Change Detection System for Toxic Molecules</h2>
<p>Once our system is tuned and is working, it can be modified to act as a fold-change detector for toxic molecules. By replacing the pGEV promoter in front of the rtTA gene with a promoter that is inducible by a toxic molecule, the amount of rtTA produced will be directly correlated to the concentration of the toxic molecule in the cellular environment.</p>
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<img src="images/figures/networkdesign-applications.png" alt="Type-1 incoherent feed-forward loop"/>
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<p>This detector system is advantageous in that it detects fold-changes rather than changes in absolute values, which gives meaning to a signal in reference to the background signal level. This allows for the network to respond only to a signal that rises significantly above the background noise.</p>
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<h1>This is not the page you're looking for.</h1>
<p>Move along.</p>
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<h1>This is not the page you're looking for.</h1>
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