Difference between revisions of "Team:Nanjing-China/Results"

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  <ul><li><a href="#https://2017.igem.org/Team:Nanjing-China/Design">Design</a></li></ul></div>
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     <p align="left" style="font-size:103%; font-family:'Courier New', Courier, monospace; z-index:3;">In the part of lab work, we have <a href="https://2017.igem.org/Team:Nanjing-China/Design">designed</a> three biosensor <a href="https://2017.igem.org/Team:Nanjing-China/Parts">sequence</a> and <a href="https://2017.igem.org/Team:Nanjing-China/PP">improved</a> an old part, <a href="http://parts.igem.org/Part:BBa_J23000">J23000</a>. What's more, all the three design have been <a href="https://2017.igem.org/Team:Nanjing-China/Demonstrate">demonstrate</a> by us.  </p>
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     <p align="left" style="font-size:105%; font-family:'Courier New', Courier, monospace; z-index:3; width:80%;">In the part of lab work, we have <a href="https://2017.igem.org/Team:Nanjing-China/Design">designed</a> three biosensor <a href="https://2017.igem.org/Team:Nanjing-China/Parts">sequence</a> and <a href="https://2017.igem.org/Team:Nanjing-China/PP">improved</a> an old part, <a href="http://parts.igem.org/Part:BBa_J23000">J23000</a>. What's more, all the three design have been <a href="https://2017.igem.org/Team:Nanjing-China/Demonstrate">demonstrate</a> by us.  </p>
 
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Revision as of 10:29, 29 October 2017

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In the part of lab work, we have <a href="https://2017.igem.org/Team:Nanjing-China/Design">designed</a> three biosensor <a href="https://2017.igem.org/Team:Nanjing-China/Parts">sequence</a> and <a href="https://2017.igem.org/Team:Nanjing-China/PP">improved</a> an old part, <a href="http://parts.igem.org/Part:BBa_J23000">J23000</a>. What's more, all the three design have been <a href="https://2017.igem.org/Team:Nanjing-China/Demonstrate">demonstrate</a> by us. 

           <img src="T-Nanjing-China-project-ch2o.png" width="35%" />

We have designed a formaldehyde sensor sequence, which is a part of our team .

The sequence is composed of PfrmR, gene frmR, flag tag, PfrmAB, gene RFP.

When the pathway works, we can see that the E.coli turns to red with naked eyes at the presence of formaldehyde.

 
<img src="T-Nanjing-China-ch2o-2.png" width="500" height="75" />

 

In order to demonstrate the design, a lot of experiments have been done.
<img src="T-Nanjing-China-ch2o-8.png" width="400" height="286" />

Figure 3. Influence of Formaldehyde Induce Time on Fluorescence Expression

  
<img src="T-Nanjing-China-ch2o-l2.png" width="600" />

Figure6. Optical density(600nm) of (a) Escherichia coli BL21 and (b) recombinant bioluminescent Escherichia coli BL21 harboring frmR-RFP fusion after 10 hours’ incubation with 800uM different aldehydes
<img src="T-Nanjing-China-ch2o-l3.png" width="350" />

Figure8.Fluorescence test of various aldehydes using recombinant bioluminescent Escherichia coli BL21
<img src="T-Nanjing-China-ch2o-l4.png" width="350" />

Figure9.The tolerance of recombinant bioluminescent Escherichia coli BL21 to various concentration of formaldehyde
It is worth to be mentioned that the team OUC help us demonstrate the result.
         <img src="T-Nanjing-China-project-h2s.png" width="40%"/>

As to the hydrogen sulfide sensor, we also designed a whole-cell biocatalytic system, displaying the concentration of hydrogen sulfide by the compound’s influence on specific genes’ expression in modified E.coli. 

In our design, we use red fluorescent protein as the indicator.When hydrogen sulfide exits, the gene transcription is activated, and the bacteria turns red.
<img src="T-Nanjing-China-h2s-2.png" width="600" height="90" />
In the experiment, we proved that the sequence worked well and was useful to detect hydrogen sulfide
<img src="T-Nanjing-China-h2s-5.png" width="400" />

Figure1.Whole-cell sequence dual-enzyme digestion

a)

<img src="T-Nanjing-China-h2s-6.png" width="400" />

b)

<img src="T-Nanjing-China-h2s-8.png" width="400" />

</td> 

           </tr>
<p>c)</p><img src="T-Nanjing-China-h2s-9.png" width="400" /><p></p></p></td> <p>Figure 2.a)RFP responsiveness of the detector system.
           b) A visible photograph of a).

           c) Test of selectivity.</p></td>
           </tr>
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     </div>

          <img src="T-Nanjing-China-project-h2.png" width="30%"/>

There is a composite of hydrogen sensor full length sequence.

The order of the elements is: HoxA-HoxB-HoxC-HoxJ-terminator-HoxP-EGFP. The sequence of HoxABCJP comes from Ralstonia eutropha H16 megaplasmid pHG1. The hole sequence acts as an hydrogen sensor.

When the amount of hydrogen goes to a higher level, Fluorescence intensity increases apparently.

               <img src="T-Nanjing-China-h2-2.png" width="650" height="100" />

The sequence was a good detecter in the lab work.

               <img src="T-Nanjing-China-h2-7.png" width="350" height="333" />

Figure1. Coomassie Brilliant Blue R-250-stained SDS-Page analysis of recombinant E.coli expressing hoxABCJ-terminator-hoxp-gfp
<img src="T-Nanjing-China-h2-8.png" width="350" height="411" />

Fingure 2. Western blot analysis of recombinant E.coli expressing his-hoxA

Fluorescence intensity remains stationary when IPTG is added.

              And  Fluorescence intensity increases in a low hydrogen atmosphere.
When the amount of hydrogen goes to a higher level Fluorescence intensity increases apparently. meaning the designed reporter pathway have worked.

<img src="T-Nanjing-China-h2-9.png" width="450" height="250" />

Figure 3. Influence of H2 concentration on fluorescence expression

 
 </div>
<p>Address</p> 
           <p>Life Science Department

             #163 Xianlin Blvd, Qixia District

             Nanjing University

             Nanjing, Jiangsu Province

             P.R. of China

             Zip: 210046
           </p>
           <p>Email</p>
           <p>nanjing_china@163.com</p>
           <p>Social media</p>
           <p>Twitter button: 

           <a href="https://twitter.com/Guja1501">iGEM Nanjing-China@nanjing_china</a></p>
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