Difference between revisions of "Team:XMU-China/Model"

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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2016.igem.org/Judging/Awards#SpecialPrizes">Best Model award</a>. </p>
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<h1 style="margin-bottom: 0px;padding-bottom:0;border-bottom: 1px solid #333;
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padding-left:16px; color:#333; font-family:"Times New Roman", Times, serif;overflow:visible; ">Proof of Concept</h1>
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                <li id="sidehead">Proof of Concept</li>
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                <li class="active"><a href="#section-1" >Experiment</a></li>
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                <li><a href="#section-2" >Result</a></li>
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                <li><a href="#section-3">Responder test</a></li>
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                  <li style="font-size:20px;color:#8968CD;border-bottom: 0.5px solid #aaa;width:95%;">Proof Of Concept</li>
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                <li ><a style="color:#aaa;" href="#section-1" >Experiment</a></li>
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                <li><a style="color:#aaa;" href="#section-2" >Result</a></li>
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                <li><a style="color:#aaa;" href="#section-3">Responder test</a></li>
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                <li><a style="color:#aaa;" href="#section-4">Reference</a></li>
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<h1 id="section-1"; style="border-bottom: 1px solid #aaa;color: #8968CD;text-shadow: 0 0 1px black;margin-bottom:.6em;padding-top: 0;
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    padding-bottom: -5%;">Experiment
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</h1>
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<h3>Entire ciruct test</h3>
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<img src="https://static.igem.org/mediawiki/2016/5/5b/T--XMU-China--result_CG_.png" width="100%;" align="center"; style="margin-bottom:20px;"/>
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<p>In our experiment, for testing our circuit more conveniently, we use the <em>cfp</em> gene and <em>gfp</em> gene to replace the responder and lysis respectly when constructing our gene circuit. Because the fluorescence is easier to be measured. Also, the strength of the fluorescence can stand for the strength of the corresponding genes' expression.</p>
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<p>When AHL around the engineered bacteria is at low concentration, AHL can't be sensed by the Lux sensor part, and the <em>cI</em> gene is expressed at the wild-type levels. While lacI-2 and YFP are repressed since the CI protein is high. When AHL around the engineered bacteria reach a certain concentration, AHL can be sensed by the sensor part,. So the promoter LuxpR is activated, CFP and lacI-1 are expressed. So the CFP protein can be tested by fluorescence measurement. And the lacI-1' product-LacR protein can inhibit the expression of <em>cI</em> gene. As a result, lacI-2 and GFP can express. The LacI-2' product can further repress <em>cI</em> gene'  expression, and GFP protein will be measured by fluorescene detection to represent the lysis gene's expression. The time interval of firstly detecting CFP' fluorescence and firstly detecting GFP' fluorescence can represent the delay time which the switch give for responder' expression.</p>
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<h1 id="section-2"; style="border-bottom: 1px solid #aaa;color: #8968CD;text-shadow: 0 0 1px black;margin-bottom:.6em;padding-top: 0;
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    padding-bottom: -5%;">Result
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</h1>
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<p>From the Last September to October, nearly one year of learning and working, we made so many of mistakes in lab work but eventually obtained ideal data and results. From the results we had, we made following analysis of our system.</p>
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<h3>1. The expression of our gene circuits</h3>
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<p>The expression of our gene circuits could be induced by AHL. When AHL is sensed by the sensor part, the promoter LuxpR is activated, CFP and lacI-1 are expressed.</p>
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<p>The <em>E. coli</em> containing our gene circuits were divided into experimental group and blank control group. We added AHL into experimental group and measured OD-600 of the <em>E. coli</em> and the fluorescence intensity of CFP and GFP for 900 minutes. The OD-600 showed the growth trend of the <em>E. coli</em>. The fluorescence intensity data and time showed the relationship of the expression of CFP and GFP to time.</p>
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<img src="https://static.igem.org/mediawiki/2016/7/7e/T--XMU-China--result_fig1_1_.png" width="100%;" align="center"; style="margin-bottom:20px;"/>
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<p><center><strong>Figure 1.1</strong> The OD-600 of <em>E. coli</em> and its variety with time on the AHL of 20ng/&mu;L and 0ng/&mu;L.</center></p>
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<p>The growth curves were similar. It was suggested that they had the same growth trend and the difference of fluorescence intensity was mainly from the expression of circuits.</p>
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<img src="https://static.igem.org/mediawiki/2016/f/fa/T--XMU-China--result_fig1_2_.png" width="100%;" align="center"; style="margin-bottom:20px;"/>
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<p><center><strong>Figure 1.2</strong> The mean CFP and GFP intensity in <em>E. coli</em> and its variety with time on the AHL of 20ng/&mu;L and 0ng/&mu;L.</center></p>
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<p>The experimental group and control group all were significantly different. The fluorescence intensity between the two groups were compared with independent t-test. It showed that the cyan and green fluorescence intensity in experimental group started to be different from that in control group in the 240th minute and 420th minute separately. The expression time of GFP was delayed to 180mins post CFP.</p>
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<h3>2. AHL Gradient Induction</h3>
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<p>We found that the concentration of AHL could have an effect on the expression of our circuits, in order to find out the relation, we decided to make a AHL gradient induction for our gene circuits. As shown in the graph, the fluorescence intensity data and time on the AHL of different concentrations show the the relationship of AHL to the expression of our circuits.</p>
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<img src="https://static.igem.org/mediawiki/2016/8/8b/T--XMU-China--result_fig1_3_.png" width="100%;" align="center"; style="margin-bottom:20px;"/>
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<p><center><strong> Figure 1.3 </strong>The mean CFP intensity in <em>E. coli</em> and its variety with time on the AHL of 1ng/&mu;L, 10ng/&mu;L, 20ng/&mu;L and 0ng/&mu;L.</center></p>
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<p>AHL would influenced the expression of CFP directly. The higher the concentration of AHL was, the more CFP expressed in a range from 1ng/&mu;L to 10ng/&mu;L. In addition, the expression time of CFP was different. T-test showed that CFP in 10ng/&mu;L and 20ng/&mu;L group started to express in the 240th minute and it started to express in the 480th minute in 1ng/&mu;L group.</p>
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<img src="https://static.igem.org/mediawiki/2016/3/37/T--XMU-China--result_fig1_4_.png" width="100%;" align="center"; style="margin-bottom:20px;"/>
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<p><center><strong> Figure 1.4 </strong>The mean GFP intensity in <em>E. coli</em> and its variety with time on the AHL of 1ng/&mu;L, 10ng/&mu;L, 20ng/&mu;L and 0ng/&mu;L.</center></p>
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<p>The concentration of AHL didn't influence the expression of GFP directly. The GFP in 10ng/&mu;L and 20ng/&mu;L group started to express in the 420th minute and it had no significant difference in expression level. The GFP didn’t express in 1ng/&mu;L group.</p>
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<h1 id="section-3"; style="border-bottom: 1px solid #aaa;color: #8968CD;text-shadow: 0 0 1px black;margin-bottom:.6em;padding-top: 0;
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    padding-bottom: -5%;">Responder test
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</h1>
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<h1 id="section-4"; style="border-bottom: 1px solid #aaa;color: #8968CD;text-shadow: 0 0 1px black;margin-bottom:.6em;padding-top: 0;
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    padding-bottom: -5%;">Reference
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</h1>
  
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<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
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<a href="http://www.xmu.edu.cn/" target="_blank"><img src="https://static.igem.org/mediawiki/2016/d/de/T--XMU-China--xmulogo_and_.png" style="width: 9%; display: inline-block; margin-left: 6%;margin-top:-6%;" /></a>
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<p style="font-size: 16px; color: #fff; border-bottom: 1px solid #fff; padding-bottom: 3%; margin-bottom: 0px;">CONTACT US</p>
  
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<a href="https://www.facebook.com/IGEM-XMU-China-1067638406629389" target="_blank" style="text-decoration:none;"><img src="https://static.igem.org/mediawiki/2016/f/f2/T--XMU-China--facebook_and_.png" style="width: 6%;margin-top: 2%;  display: inline-block;" /><span style=" padding-left:4%;font-size: 14px; color: #fff; margin-top: 10%; margin-bottom: 0px;">https://www.facebook.com/IGEM-XMU-China-1067638406629389</a></span><br/>
  
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<a href="https://www.twitter.com/Amoy_igem" target="_blank" style="text-decoration:none;"><img src="https://static.igem.org/mediawiki/2016/5/5a/T--XMU-China--twitter_and_.png" style="width: 6%; display: inline-block; margin-top: 2%;" />
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<h2> Modeling</h2>
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<span style="padding-left:3%;font-size: 14px; color: #fff; margin-top: 10%; margin-bottom: 0px;">https://www.twitter.com/Amoy_igem</span></a><br/>
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<p>Mathematical models and computer simulations provide a great way to describe the function and operation of BioBrick Parts and Devices. Synthetic Biology is an engineering discipline, and part of engineering is simulation and modeling to determine the behavior of your design before you build it. Designing and simulating can be iterated many times in a computer before moving to the lab. This award is for teams who build a model of their system and use it to inform system design or simulate expected behavior in conjunction with experiments in the wetlab.</p>
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<img src="https://static.igem.org/mediawiki/2016/e/ea/T--XMU-China--Interlab-gmail_and_.png" style="width:6%; display: inline-block;margin-top: 2%;" />
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<span style="font-size: 14px; color: #fff; margin-top: 10%; margin-bottom: 0px;padding-left:3%;">igemxmu@gmail.com</span>
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<h5> Inspiration </h5>
 
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<p>
 
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Here are a few examples from previous teams:
 
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</p>
 
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<ul>
 
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<li><a href="https://2014.igem.org/Team:ETH_Zurich/modeling/overview">ETH Zurich 2014</a></li>
 
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<li><a href="https://2014.igem.org/Team:Waterloo/Math_Book">Waterloo 2014</a></li>
 
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Revision as of 05:46, 14 October 2016

Proof of Concept

Appropriate attribution is half of success.

Experiment

Entire ciruct test

In our experiment, for testing our circuit more conveniently, we use the cfp gene and gfp gene to replace the responder and lysis respectly when constructing our gene circuit. Because the fluorescence is easier to be measured. Also, the strength of the fluorescence can stand for the strength of the corresponding genes' expression.

When AHL around the engineered bacteria is at low concentration, AHL can't be sensed by the Lux sensor part, and the cI gene is expressed at the wild-type levels. While lacI-2 and YFP are repressed since the CI protein is high. When AHL around the engineered bacteria reach a certain concentration, AHL can be sensed by the sensor part,. So the promoter LuxpR is activated, CFP and lacI-1 are expressed. So the CFP protein can be tested by fluorescence measurement. And the lacI-1' product-LacR protein can inhibit the expression of cI gene. As a result, lacI-2 and GFP can express. The LacI-2' product can further repress cI gene' expression, and GFP protein will be measured by fluorescene detection to represent the lysis gene's expression. The time interval of firstly detecting CFP' fluorescence and firstly detecting GFP' fluorescence can represent the delay time which the switch give for responder' expression.

Result

From the Last September to October, nearly one year of learning and working, we made so many of mistakes in lab work but eventually obtained ideal data and results. From the results we had, we made following analysis of our system.

1. The expression of our gene circuits

The expression of our gene circuits could be induced by AHL. When AHL is sensed by the sensor part, the promoter LuxpR is activated, CFP and lacI-1 are expressed.

The E. coli containing our gene circuits were divided into experimental group and blank control group. We added AHL into experimental group and measured OD-600 of the E. coli and the fluorescence intensity of CFP and GFP for 900 minutes. The OD-600 showed the growth trend of the E. coli. The fluorescence intensity data and time showed the relationship of the expression of CFP and GFP to time.

Figure 1.1 The OD-600 of E. coli and its variety with time on the AHL of 20ng/μL and 0ng/μL.

The growth curves were similar. It was suggested that they had the same growth trend and the difference of fluorescence intensity was mainly from the expression of circuits.

Figure 1.2 The mean CFP and GFP intensity in E. coli and its variety with time on the AHL of 20ng/μL and 0ng/μL.

The experimental group and control group all were significantly different. The fluorescence intensity between the two groups were compared with independent t-test. It showed that the cyan and green fluorescence intensity in experimental group started to be different from that in control group in the 240th minute and 420th minute separately. The expression time of GFP was delayed to 180mins post CFP.

2. AHL Gradient Induction

We found that the concentration of AHL could have an effect on the expression of our circuits, in order to find out the relation, we decided to make a AHL gradient induction for our gene circuits. As shown in the graph, the fluorescence intensity data and time on the AHL of different concentrations show the the relationship of AHL to the expression of our circuits.

Figure 1.3 The mean CFP intensity in E. coli and its variety with time on the AHL of 1ng/μL, 10ng/μL, 20ng/μL and 0ng/μL.

AHL would influenced the expression of CFP directly. The higher the concentration of AHL was, the more CFP expressed in a range from 1ng/μL to 10ng/μL. In addition, the expression time of CFP was different. T-test showed that CFP in 10ng/μL and 20ng/μL group started to express in the 240th minute and it started to express in the 480th minute in 1ng/μL group.

Figure 1.4 The mean GFP intensity in E. coli and its variety with time on the AHL of 1ng/μL, 10ng/μL, 20ng/μL and 0ng/μL.

The concentration of AHL didn't influence the expression of GFP directly. The GFP in 10ng/μL and 20ng/μL group started to express in the 420th minute and it had no significant difference in expression level. The GFP didn’t express in 1ng/μL group.

Responder test

Reference

Name: XMU-China School: Xiamen University


Address: Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P. R. China 361005

Name: XMU-China School: Xiamen University


Address: Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P. R. China 361005