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

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             <div style="position:relative; top:-40px; z-index:3;"><img src="https://static.igem.org/mediawiki/2017/f/f1/T-Nanjing-China-project-ch2o.png" width="35%" /></div>
 
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               <td colspan="2"><p>We have designed a formaldehyde sensor sequence, which is a part of our team .</p>
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               <td colspan="2"><p>As  is shown to all of us, the whole sequence is about 1500 base-pairs while the  vector is 2000 base-pairs. SDS-PAGE analysis also showed the expression of the  regulator, protein FRMR, around 15kd. Therefore, we moved forward to further  property study.</p></td>
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                <p>The sequence is composed of PfrmR, gene frmR, flag tag, PfrmAB, gene RFP.</p>
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                <p>When the pathway works, we can see that the E.coli turns to red with naked eyes at the presence of formaldehyde. </p>
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                <blockquote>
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                  <p>&nbsp;</p>
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              </blockquote></td>
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               <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/b/b4/T-Nanjing-China-ch2o-2.png" width="500" height="75" /></div></td>  
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                <p><img src="https://static.igem.org/mediawiki/2017/c/ca/T-Nanjing-China-ch2o-6.png" width="310" height="403" /></p>
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                <p><font size="-1">Figure1.Whole-cell sequence dual-enzyme digestion</font></p>
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              </div></td>
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              <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/a/ae/T-Nanjing-China-ch2o-7.png" width="400" height="366" />
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              <p><font size="-1">Figure2.SDS-PAGE analysis of recombinant E.coli expressing FrmR</font></p></div></td>
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             <td colspan="2"><blockquote>
 
             <td colspan="2"><blockquote>
 
               <p>&nbsp;</p>
 
               <p>&nbsp;</p>
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               <p>In order to demonstrate the design, a lot of experiments have been done.</p>
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               </blockquote>
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            </blockquote></td>
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              <p>This diagram illustrates the fluorescence intensity change induced by formaldehyde along with interval time 2 hours. The peak value occurs after 6 hours, that is, only requiring 6 hours, the detecting results can be seen with naked-eyes. Compared to the blank control, experimental group with formaldehyde induction turns to pink apparently, meaning the designed reporter pathway have worked. </p></td>
 
             </tr>
 
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               <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/8/88/T-Nanjing-China-ch2o-8.png" width="400" height="286" /><p><font size="-1">Figure  1. Influence of Formaldehyde Induce Time on Fluorescence Expression</font></p></div></td>
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               <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/8/88/T-Nanjing-China-ch2o-8.png" width="350" height="251" /><p><font size="-1">Figure  3. Influence of Formaldehyde Induce Time on Fluorescence Expression</font></p></div></td>
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               <td>&nbsp;</td>
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                <p><img src="https://static.igem.org/mediawiki/2017/0/04/T-Nanjing-China-ch2o-9.png" width="350" height="245" /></p>
 +
              <p><font size="-1">Figure4.A photograph of E.coli cells containing the formaldehyde-induced RFP expression  plasmid, or without formaldehyde induction, and re-suspended in PBS  buffer(pH7.4)</font></p>
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            <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/7/76/T-Nanjing-China-ch2o-l2.png" width="600" />
 
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            <p><font size="-1">Figure2. 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</font></p></div></td>
 
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               <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/6/6a/T-Nanjing-China-ch2o-l3.png" width="350" />
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               <td colspan="2"><p>&nbsp;</p>
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            <p><font size="-1">Figure3.Fluorescence test of various aldehydes using recombinant bioluminescent Escherichia coli BL21</font></p></div></td>
+
                <p>Moreover, in order to set up the corresponding relationship between the quantity of formaldehyde and the fluorescence value, we prepared a series of gradient concentrations of formaldehyde. We found out that from the concentrations of 300 micromole to 600 micromole, a preferable equation of linear regression could be obtained, which laid the cornerstone for creating precise and sensitive detecting devices.
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              <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/2/22/T-Nanjing-China-ch2o-l4.png" width="350" />
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              </p></td>
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            <p><font size="-1">Figure4.The tolerance of recombinant bioluminescent Escherichia coli BL21 to various concentration of formaldehyde</font></p></div></td>
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              <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/7/76/T-Nanjing-China-ch2o-10.png" width="350" height="265" /></div></td>
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              <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/9/98/T-Nanjing-China-ch2o-11.png" width="350" height="265" /></div></td>
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            </tr>
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            <td colspan="2"><p align="center"><font size="-1">Figure5.Fluorescence measurement of E.coli cells containing the formaldehyde-induced RFP expression plasmid after gradient concentrations of formaldehyde induction and re-suspended in PBS buffer(pH7.4)</font></p></td>
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          </tr>
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            <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/7/76/T-Nanjing-China-ch2o-l2.png" width="600" />
 +
            <p><font size="-1">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</font></p></div></td></tr>
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            <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/0/0e/T-Nanjing-China-ch2o-l1.png" width="600" />
 +
            <p><font size="-1">Figure7.Response growth curve for recombinant bioluminescent Escherichia coli BL21 to different concentration of formaldehyde</font></p></div></td></tr>
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            <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/6/6a/T-Nanjing-China-ch2o-l3.png" width="350" />
 +
            <p><font size="-1">Figure8.Fluorescence test of various aldehydes using recombinant bioluminescent Escherichia coli BL21</font></p></div></td>
 +
            <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/2/22/T-Nanjing-China-ch2o-l4.png" width="350" />
 +
            <p><font size="-1">Figure9.The tolerance of recombinant bioluminescent Escherichia coli BL21 to various concentration of formaldehyde</font></p></div></td></tr>
 
           <tr>
 
           <tr>
 
             <td colspan="2">It is worth to be mentioned that the team <a href="https://2017.igem.org/Team:Nanjing-China/Collaborations">OUC</a> help us demonstrate the result.</td></tr>
 
             <td colspan="2">It is worth to be mentioned that the team <a href="https://2017.igem.org/Team:Nanjing-China/Collaborations">OUC</a> help us demonstrate the result.</td></tr>
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            <td colspan="2"><p>As to the hydrogen sulfide sensor, we also designed a whole-cell biocatalytic system, displaying the concentration of hydrogen sulfide by the compound&rsquo;s influence on specific genes&rsquo; expression in modified E.coli. </p>              <p>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.</p></td>
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            <td colspan="2"><p>A plate sensitive assay measuring S2– tolerance of E. coli cells with constructed probe pathway. All plates were incubated at 37℃ for 18 h before being read. No significant influence appeared to the growth of E. coli at a concentration lower than 10mmol/L.</p></td>
 
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               <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/d/d8/T-Nanjing-China-h2s-2.png" width="600" height="90" /></div></td>
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               <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/3/3c/T-Nanjing-China-h2s-4-1.jpg" width="524" height="400" /></div></td>
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              <td colspan="2">In the experiment, we proved that the sequence worked well and was useful to detect hydrogen sulfide</td>
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           <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/c/c6/T-Nanjing-China-h2s-5.png" width="400" />
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           <td><p>We analysised the product by dual-enzyme digestion and electrophoresis.</p></td>
 +
          <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/c/c6/T-Nanjing-China-h2s-5.png" width="400" />
 
             <p><font size="-1">Figure1.Whole-cell sequence dual-enzyme digestion</font></p></div></td>
 
             <p><font size="-1">Figure1.Whole-cell sequence dual-enzyme digestion</font></p></div></td>
 
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             <td><p><font size="-1">a)</font></p><img src="https://static.igem.org/mediawiki/2017/6/63/T-Nanjing-China-h2s-6.png" width="400" />
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             <td><p><font size="-1">a)</font></p><img src="https://static.igem.org/mediawiki/2017/6/63/T-Nanjing-China-h2s-6.png" width="350" /></td>
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            </td>
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             <td><p><font size="-1">b)</font></p><img src="https://static.igem.org/mediawiki/2017/4/4d/T-Nanjing-China-h2s-8.png" width="350" /><p><font size="-1"></td>
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             <td><p><font size="-1">b)</font></p><div align="center"><img src="https://static.igem.org/mediawiki/2017/4/4d/T-Nanjing-China-h2s-8.png" width="400" /><p><font size="-1"></td>
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             <td><p><font size="-1">c)</font></p><img src="https://static.igem.org/mediawiki/2017/b/bc/T-Nanjing-China-h2s-9.png" width="400" /><p><font size="-1"></p></p></td>
 
             <td><p><font size="-1">c)</font></p><img src="https://static.igem.org/mediawiki/2017/b/bc/T-Nanjing-China-h2s-9.png" width="400" /><p><font size="-1"></p></p></td>
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             <td><p><font size="-1">Figure  2.a)RFP responsiveness of the detector system.<br />
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             <td><p><font size="-1">Figure2.a)RFP responsiveness of the detector system.<br />
 
             b) A visible photograph of a).<br />
 
             b) A visible photograph of a).<br />
 
             c) Test of selectivity.</font></p></td>
 
             c) Test of selectivity.</font></p></td>
 +
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            <td colspan="2"><p>RFP responsiveness of the detector system. Cells were grown to midlog phase under aerobic conditions and 0 ~ 250 μM Na2S. Cells were harvest after 17h and assayed for fluorescence intensity. Error bars indicate SD of the mean.</p></td>
 
             </tr>
 
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           <img src="https://static.igem.org/mediawiki/2017/0/09/T-Nanjing-China-project-h2.png" width="30%"/>
 
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              <p>There is a composite of hydrogen sensor full length sequence. </p>
 
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              <p>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.</p>
 
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              <p>When the amount of hydrogen goes to a higher level, Fluorescence intensity increases apparently.</p>
 
−
              </td></tr>
 
 
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               <td colspan="2"><p>Now  we&rsquo;ve succefully detected the  protein expression by SDS-Page analysis and Western blot analysis.</p></td>
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                <img src="https://static.igem.org/mediawiki/2017/b/b5/T-Nanjing-China-h2-2.png" width="650" height="100" />
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              <p>The sequence was a good detecter in the lab work.</p>
 
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               <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/6/69/T-Nanjing-China-h2-9.png" width="500" height="250" />
 
               <p><font size="-1">Figure  3. Influence of H2  concentration on fluorescence expression</font></p>
 
               <p><font size="-1">Figure  3. Influence of H2  concentration on fluorescence expression</font></p>
 
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Revision as of 00:30, 1 November 2017

Team:Nanjing-China - 2017.igem.org

 

In the part of lab work, we have designed three biosensor sequence and improved an old part, J23000. What's more, all the three design have been demonstrate by us.

 

As is shown to all of us, the whole sequence is about 1500 base-pairs while the vector is 2000 base-pairs. SDS-PAGE analysis also showed the expression of the regulator, protein FRMR, around 15kd. Therefore, we moved forward to further property study.

Figure1.Whole-cell sequence dual-enzyme digestion

Figure2.SDS-PAGE analysis of recombinant E.coli expressing FrmR

 

This diagram illustrates the fluorescence intensity change induced by formaldehyde along with interval time 2 hours. The peak value occurs after 6 hours, that is, only requiring 6 hours, the detecting results can be seen with naked-eyes. Compared to the blank control, experimental group with formaldehyde induction turns to pink apparently, meaning the designed reporter pathway have worked.

Figure 3. Influence of Formaldehyde Induce Time on Fluorescence Expression

Figure4.A photograph of E.coli cells containing the formaldehyde-induced RFP expression plasmid, or without formaldehyde induction, and re-suspended in PBS buffer(pH7.4)

 

Moreover, in order to set up the corresponding relationship between the quantity of formaldehyde and the fluorescence value, we prepared a series of gradient concentrations of formaldehyde. We found out that from the concentrations of 300 micromole to 600 micromole, a preferable equation of linear regression could be obtained, which laid the cornerstone for creating precise and sensitive detecting devices.

Figure5.Fluorescence measurement of E.coli cells containing the formaldehyde-induced RFP expression plasmid after gradient concentrations of formaldehyde induction and re-suspended in PBS buffer(pH7.4)

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

Figure7.Response growth curve for recombinant bioluminescent Escherichia coli BL21 to different concentration of formaldehyde

Figure8.Fluorescence test of various aldehydes using recombinant bioluminescent Escherichia coli BL21

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.

A plate sensitive assay measuring S2– tolerance of E. coli cells with constructed probe pathway. All plates were incubated at 37℃ for 18 h before being read. No significant influence appeared to the growth of E. coli at a concentration lower than 10mmol/L.

We analysised the product by dual-enzyme digestion and electrophoresis.

Figure1.Whole-cell sequence dual-enzyme digestion

a)

b)

c)

Figure2.a)RFP responsiveness of the detector system.
b) A visible photograph of a).
c) Test of selectivity.

RFP responsiveness of the detector system. Cells were grown to midlog phase under aerobic conditions and 0 ~ 250 μM Na2S. Cells were harvest after 17h and assayed for fluorescence intensity. Error bars indicate SD of the mean.

Now we’ve succefully detected the protein expression by SDS-Page analysis and Western blot analysis.

Figure1. Coomassie Brilliant Blue R-250-stained SDS-Page analysis of recombinant E.coli expressing hoxABCJ-terminator-hoxp-gfp

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.

Figure 3. Influence of H2 concentration on fluorescence expression

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Life Science Department
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Nanjing University
Nanjing, Jiangsu Province
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Zip: 210046

Email

nanjing_china@163.com

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