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

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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><a href=" https://2016.igem.org/Team:XMU-China/Description">Description</a></li>
 
           <li><a href=" https://2016.igem.org/Team:XMU-China/Description">Description</a></li>
 
         <li><a href="https://2016.igem.org/Team:XMU-China/Design">Design</a></li>
 
         <li><a href="https://2016.igem.org/Team:XMU-China/Design">Design</a></li>
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         <li><a href="https://2016.igem.org/Team:XMU-China/Proof">Proof of Concept</a></li>
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         <li><a href="https://2016.igem.org/Team:XMU-China/Proof">Proof of concept</a></li>
 
         <li><a href="https://2016.igem.org/Team:XMU-China/Demonstrate">Demonstrate</a></li>
 
         <li><a href="https://2016.igem.org/Team:XMU-China/Demonstrate">Demonstrate</a></li>
 
         <li><a href="https://2016.igem.org/Team:XMU-China/Results">Results</a></li>
 
         <li><a href="https://2016.igem.org/Team:XMU-China/Results">Results</a></li>
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                 <li id="sidehead">Proof Of Concept</li>
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                 <li id="sidehead">Results</li>
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                 <li class="active"><a href="#section-1" >Background</a></li>
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                 <li class="active"><a href="#section-1" >Experiment</a></li>
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                 <li><a href="#section-2" >Abstract</a></li>
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                 <li><a href="#section-2" >Result</a></li>
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                 <li><a href="#section-3">Reference</a></li>
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                 <li><a href="#section-3">Responder test</a></li>
 +
                <li><a href="#section-4">Reference</a></li>  
 
                
 
                
 
             </ul>
 
             </ul>
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                   <li style="font-size:20px;color:#8968CD;border-bottom: 0.5px solid #aaa;width:95%;">Proof Of Concept</li>
 
                   <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" >Background</a></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" >Abstract</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">Reference</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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  <div class="col-md-10" >
  
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<h1 style="border-bottom: 1px solid #aaa;color: #8968CD;text-shadow: 0 0 1px black;margin-bottom:.6em;padding-top: 0;
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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%;">Background
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     padding-bottom: -5%;">Experiment
 
</h1>
 
</h1>
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<p>Bacteria are the main pathogen of human communicable disease during the past years. In 14 century, The Black Death plague caused by Yersinia pestis led over 75 million people death in the world, and Cholera caused by Vibrio cholera became global pandemic for eight times. In 1907, Typhoid bacillus was found the the arch-criminal of Typhoid Mary event. However, the table was turned by the discovery of penicillin in 1928. And new category of antibiotics had been discovered rapidly during the following years. Antibiotic therapy has saved countless lives. </p>
+
<h3>Entire ciruct test</h3>
 +
<img src="https://static.igem.org/mediawiki/2016/5/5b/T--XMU-China--result_CG_.png" width="100%;" align="center"; style="margin-bottom:20px;"/>
 +
<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>
  
−
<p>But for the abuse of antibiotics, in 21th century, a spreading of drug resistance appears. When the antibiotics kill pathogenic bacteria, some of them mutating and developing the "drug-resistance". Drug resistant bacteria flourish in the absence of diminished competition, and the resistance can be transferred among the bacteria, which made their infections considerable mortality and morbidity [1]. Furthermore, the prevalence of the infections is still increasing by abused antibiotics and resistance spreading. It is predicted that 10 million people would be killed for the drug-resistant pathogen infections each year by 2050 [2]. Multidrug-resistant (MDR) pathogens has been identified as one of the top three threats to human health by the World Health Organization (WHO) [3]. </p>
+
<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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<p id="section-2">Bacteria develop clinically significant resistance in a period of just months to years. The new antimicrobials or modifications of current arsenal may not address the trends in resistance effectively [3]. Combination therapies for the treatment are needed urgently [1].</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;
 +
    padding-bottom: -5%;">Result
 +
</h1>
 +
<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>
  
−
<h1 style="border-bottom: 1px solid #aaa;color: #8968CD;text-shadow: 0 0 1px black;margin-bottom:.6em;padding-top: 0;
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<h3>1. The expression of our gene circuits</h3>
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     padding-bottom: -5%;">Abstract
+
 
 +
<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>
 +
<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>
 +
 
 +
<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;"/>
 +
<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>
 +
<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>
 +
 
 +
<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;"/>
 +
<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>
 +
 
 +
<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>
 +
 
 +
<h3>2. AHL Gradient Induction</h3>
 +
 
 +
<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>
 +
 
 +
<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;"/>
 +
<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>
 +
 
 +
<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>
 +
 
 +
<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;"/>
 +
<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>
 +
 
 +
<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>
 +
 
 +
 
 +
<h1 id="section-3"; style="border-bottom: 1px solid #aaa;color: #8968CD;text-shadow: 0 0 1px black;margin-bottom:.6em;padding-top: 0;
 +
     padding-bottom: -5%;">Responder test
 
</h1>
 
</h1>
−
<p>Our team supposed to construct programmable bacteria for combating specific pathogens. Following the theory of “input to render-loop to output”, we designed two models. Model 1 can be divided into four parts: sensor, killer, toggle switch and lysis. We performed it by constructing a gene circuit which can detect the gram-negative bacteria and release toxins to kill the target bacteria. Model 2 was composed of three parts: sensor, killer and self-destruction switch. We built one circuit for the model examination, by <span id="section-3">which</a> the engineered bacterium can lyse synchronously and release M13 phages. Multiple functional components of each model lead varies functions of the whole circuit when the three parts assembled. </p>
 
  
−
<h1 style="border-bottom: 1px solid #aaa;color: #8968CD;text-shadow: 0 0 1px black;margin-bottom:.6em;padding-top: 0;
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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;
 
     padding-bottom: -5%;">Reference
 
     padding-bottom: -5%;">Reference
 
</h1>
 
</h1>
−
<p>[1] Brooks, B. D., & Brooks, A. E. (2014). Therapeutic strategies to combat antibiotic resistance. Advanced Drug Delivery Reviews, 78(C), 14–27. <br/>
+
 
−
[2] McCullough, A. R., Parekh, S., Rathbone, J., Del Mar, C. B., & Hoffmann, T. C. (2016). A systematic review of the public's knowledge and beliefs about antibiotic resistance. Journal o Antimicrobial Chemotherapy, 71(1), 27–33.<br/>
+
−
[3] Worthington, R. J., & Melander, C. (2013). Combination approaches to combat multidrug-resistant bacteria. Trends in Biotechnology, 31(3), 179–186.
+
−
</p>
+
 
</div>
 
</div>
 
</div>
 
</div>

Revision as of 03:05, 14 October 2016

Results

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