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− | <ul><li><a href="https://2017.igem.org/Team:Nanjing-China/ | + | <ul><li><a href="https://2017.igem.org/Team:Nanjing-China/Design">Design</a></li></ul></div> |
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+ | <ul><li><a href="https://2017.igem.org/Team:Nanjing-China/Notebook">Notebook</a></li></ul></div> | ||
+ | <div class="sub"> | ||
+ | <ul><li><a href="https://2017.igem.org/Team:Nanjing-China/Demonstrate">Results</a></li></ul></div> | ||
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+ | <div class="sub"> | ||
+ | <ul><li><a href="https://2017.igem.org/Team:Nanjing-China/Demonstrate">Demonstrate</a></li></ul></div> | ||
+ | <div class="sub"> | ||
+ | <ul><li><a href="https://2017.igem.org/Team:Nanjing-China/Improvement">Improvement</a></li></ul></div> | ||
</div></div> | </div></div> | ||
+ | </div> | ||
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</div> | </div> | ||
<div class="container" align="center"> | <div class="container" align="center"> | ||
− | <div class="header"><img src="https://static.igem.org/mediawiki/2017/ | + | <div class="header"><img src="https://static.igem.org/mediawiki/2017/9/97/T-Nanjing-China-title-results.png" width="100%" onload="MM_timelinePlay('Timeline2'); MM_effectAppearFade('menu', 2000, 0, 100, false)" /> |
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− | <div class="word" style=" position:relative; | + | <div class="word" style=" position:relative; background-color:#FFF;"> |
− | < | + | <p align="left" style="font-size:115%; font-family:'Courier New', Courier, monospace; z-index:3;"> </p> |
− | <p style="font-size: | + | <p align="left" style="font-size:115%; font-family:'Courier New', Courier, monospace; z-index:3;">In the part of lab work, we have designed <a href="https://2017.igem.org/Team:Nanjing-China/Parts">three biosensor sequences</a> and <a href="https://2017.igem.org/Team:Nanjing-China/PP">improved</a> an old part, BBa_J23100. What's more, all the three designs have been demonstrated by us. </p> |
+ | <p align="left" style="font-size:115%; font-family:'Courier New', Courier, monospace; z-index:3;"> </p> | ||
</div> | </div> | ||
− | + | <div id="ch2o"> | |
<div align="center"> | <div align="center"> | ||
− | <img src="https://static.igem.org/mediawiki/2017/f/f1/T-Nanjing-China-project-ch2o.png" width="35%" /> | + | <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> |
<table width="80%" border="0" cellspacing="1" cellpadding="1"> | <table width="80%" border="0" cellspacing="1" cellpadding="1"> | ||
<tr> | <tr> | ||
− | <td colspan="2"> | + | <td colspan="2"><p>As is shown in figure1, the whole sequence of our formaldehyde pathway is about 1500 base-pairs while the vector is 2000 base-pairs. SDS-PAGE analysis(figure 2) also shows the expression of the regulator, protein FRMR, around 15kd. Therefore, we moved forward to further property study.</p></td> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td | + | <td><div align="center"> |
− | + | <p><img src="https://static.igem.org/mediawiki/2017/c/ca/T-Nanjing-China-ch2o-6.png" height="400" /></p> | |
+ | <p><font size="-1">Figure1.Whole-cell sequence dual-enzyme digestion</font></p> | ||
+ | </div></td> | ||
+ | <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/a/ae/T-Nanjing-China-ch2o-7.png" height="400" /> | ||
+ | <p><font size="-1">Figure2.SDS-PAGE analysis of recombinant E.coli expressing FrmR</font></p></div></td> | ||
+ | </tr> | ||
<tr> | <tr> | ||
<td colspan="2"><blockquote> | <td colspan="2"><blockquote> | ||
− | <p> | + | <p> </p> |
+ | </blockquote> | ||
+ | <p>Figure3 illustrates the fluorescence intensity change induced by formaldehyde along with an interval of 2 hours. The peak value occurs after 6 hours, which means the detecting results can be seen with naked-eyes after only 6 hours. As is shown in figure 4, compared to the blank control, experimental group with formaldehyde induction turns to pink apparently, meaning the designed reporter pathway has worked. </p></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><div align="center" | + | <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/8/88/T-Nanjing-China-ch2o-8.png" width="500" /><p><font size="-1">Figure 3. Influence of Formaldehyde Induce Time on Fluorescence Expression</font></p></div></td> |
− | <td><div align="center"> | + | <td> |
− | + | </tr> | |
+ | <tr> | ||
+ | <td colspan="2"> <div align="center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/0/04/T-Nanjing-China-ch2o-9.png" width="500" height="353" /> | ||
<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> | <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> | ||
</div></td> | </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 validate the result.</td></tr> | ||
<tr> | <tr> | ||
+ | <td colspan="2"><p> </p> | ||
+ | <p>Moreover, in order to set up the corresponding relationship between the quantity of formaldehyde and the fluorescence value, we prepared a series of concentrations of formaldehyde(figure5a.). We found out that from the concentration of 300 micromole to 600 micromole, a preferable equation of linear regression could be obtained(figure5b.), which laid the cornerstone for creating precise and sensitive detecting devices. | ||
+ | </p></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/7/76/T-Nanjing-China-ch2o-10.png" width=" | + | <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/7/76/T-Nanjing-China-ch2o-10.png" width="500" />a)</div></td> |
− | <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/9/98/T-Nanjing-China-ch2o-11.png" width=" | + | </tr> |
+ | <tr> | ||
+ | <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/9/98/T-Nanjing-China-ch2o-11.png" width="500" />b)</div></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td colspan="2"> | + | <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> |
− | + | </tr> | |
− | </ | + | <tr> |
+ | <td colspan="2"><p > </p> | ||
+ | <p >Then we incubated our recombinant E. coli for more than 10 hours to test their reponse growth rates(figure 6a) and tolerance(figure 6b) to different concentrations of formaldehyde. </p></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td colspan="2"> | + | <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">Figure6a.Response growth curve for recombinant bioluminescent Escherichia coli BL21 to different concentration of formaldehyde</font></p></div></td></tr> | ||
+ | <tr> | ||
+ | <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/2/22/T-Nanjing-China-ch2o-l4.png" width="500" /> | ||
+ | <p><font size="-1">Figur6b.The tolerance of recombinant bioluminescent Escherichia coli BL21 to various concentration of formaldehyde</font></p></div></td></tr> | ||
+ | <tr> | ||
+ | <td colspan="2"><p> </p> | ||
+ | <p>Finally, we tested the selectivity of our formaldehyde pathway by inducing the recombinant E. coli with acetaldehyde, DMSO and 6 other aldehydes. The results(figure 7) demonstrated good selsctivity of our recombinant plasmids. </p></td></tr> | ||
+ | <tr> | ||
+ | <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/6/6a/T-Nanjing-China-ch2o-l3.png" width="500" /> | ||
+ | <p><font size="-1">Figure7.Fluorescence test of various aldehydes using recombinant bioluminescent Escherichia coli BL21</font></p></div></td></tr> | ||
</table> | </table> | ||
</div> | </div> | ||
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<img src="https://static.igem.org/mediawiki/2017/c/c7/T-Nanjing-China-project-h2s.png" width="40%"/> | <img src="https://static.igem.org/mediawiki/2017/c/c7/T-Nanjing-China-project-h2s.png" width="40%"/> | ||
<table width="80%" border="0" cellspacing="1" cellpadding="1"> | <table width="80%" border="0" cellspacing="1" cellpadding="1"> | ||
+ | <td colspan="2"><p>We first analyzed the product by dual-enzyme digestion and electrophoresis(figure8). As can be seen, our hydrogen sulfide sensing sequence is over 3,000 base pairs.</p></td></tr> | ||
<tr> | <tr> | ||
− | + | <td colspan="2"><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">Figure8.Whole-cell sequence dual-enzyme digestion</font></p></div></td> | |
− | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | + | <td colspan="2"><p> </p> | |
+ | <p>Next, we conducted a plate sensitive assay to measure the S<sup>2–</sup> tolerance of E. coli cells with constructed probe pathway. All plates were incubated at 37℃ for 18 h before reading. No significant influence appeared to the growth of E. coli at a concentration lower than 10mmol/L.</p></td> | ||
</tr> | </tr> | ||
− | + | <tr> | |
− | <td colspan="2"> | + | <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" /><br> |
+ | <font size="-1">Figure 9. Tolerance test</font></div></td> | ||
</tr> | </tr> | ||
− | + | <tr> | |
− | + | <td colspan="2"><p> </p> | |
− | + | <p>Cells were then grown to midlog phase under aerobic conditions and 0 ~ 250 μM Na<sub>2</sub>S. Cells were harvest after 17h and assayed for fluorescence intensity. Error bars indicate SD of the mean.</p></td> | |
− | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <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=" | + | <td colspan="2"><p><font size="-1">Figure10 a)RFP responsiveness of the detector system.</font></p> <div align="center"><img src="https://static.igem.org/mediawiki/2017/6/63/T-Nanjing-China-h2s-6.png" width="500" /></div></td></tr> |
− | + | <tr> | |
− | <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=" | + | <td colspan="2"><p> </p> |
+ | <p><font size="-1">Figure10 b) A visible photograph of a).</font></p> | ||
+ | <div align="center"><img src="https://static.igem.org/mediawiki/2017/4/4d/T-Nanjing-China-h2s-8.png" width="500" /></div></td> | ||
</tr> | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2"><p> </p> | ||
+ | <p>Finally, we examined the plasmid's selectivity against SO<sub>4</sub><sup>2-</sup>, SO<sub>3</sub><sup>2-</sup> and 4 other chemical reagents(figure 11).</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
<tr> | <tr> | ||
− | <td>< | + | <td colspan="2"><div align="center"> |
− | < | + | <img src="https://static.igem.org/mediawiki/2017/b/bc/T-Nanjing-China-h2s-9.png" width="500" /> |
− | + | <p><font size="-1">Figure 11.Test of selectivity.</font></p> | |
− | + | </div></td> | |
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2"><p> </p></td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
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<img src="https://static.igem.org/mediawiki/2017/0/09/T-Nanjing-China-project-h2.png" width="30%"/> | <img src="https://static.igem.org/mediawiki/2017/0/09/T-Nanjing-China-project-h2.png" width="30%"/> | ||
<table width="80%" border="0" cellspacing="1" cellpadding="1"> | <table width="80%" border="0" cellspacing="1" cellpadding="1"> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
<tr> | <tr> | ||
− | <td colspan="2"> | + | <td colspan="2"><p>First we succefully detected the protein expression by SDS-Page(figure 12a) analysis and Western blot(figure 12b) analysis.</p></td> |
− | + | ||
− | + | ||
</tr> | </tr> | ||
− | |||
− | |||
− | |||
− | |||
<tr> | <tr> | ||
<td><div align="center"> | <td><div align="center"> | ||
− | <img src="https://static.igem.org/mediawiki/2017/1/10/T-Nanjing-China-h2-7.png | + | <img src="https://static.igem.org/mediawiki/2017/1/10/T-Nanjing-China-h2-7.png" height="400" /> |
− | <p><font size="-1"> | + | <p><font size="-1">Figure12a. Coomassie Brilliant Blue R-250-stained SDS-Page analysis of recombinant E.coli expressing hoxABCJ-terminator-hoxp-gfp</font></p> |
</div></td> | </div></td> | ||
− | <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/9/9f/T-Nanjing-China-h2-8.png | + | <td><div align="center"><img src="https://static.igem.org/mediawiki/2017/9/9f/T-Nanjing-China-h2-8.png" height="400" /> |
− | <p><font size="-1">Fingure | + | <p><font size="-1">Fingure 12b. Western blot analysis of recombinant E.coli expressing his-hoxA</font></p></div></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td colspan="2">< | + | <td colspan="2"><p> </p> |
− | <p>Fluorescence intensity remains stationary when IPTG is added. | + | <p>Fluorescence intensity remains stationary when IPTG is added. |
− | + | Whereas, it increases in a low hydrogen atmosphere. When the amount of hydrogen goes to an even higher level, fluorescence intensity increases apparently, meaning the designed report pathway works as expected(figure 13). </p></td> | |
− | + | ||
− | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/6/69/T-Nanjing-China-h2-9.png" width=" | + | <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/6/69/T-Nanjing-China-h2-9.png" width="550" /> |
− | <p><font size="-1">Figure | + | <p><font size="-1">Figure 13. Influence of H<sub>2</sub> concentration on fluorescence expression</font></p> |
</div></td> | </div></td> | ||
</tr> | </tr> |
Latest revision as of 15:23, 1 November 2017
In the part of lab work, we have designed three biosensor sequences and improved an old part, BBa_J23100. What's more, all the three designs have been demonstrated by us.
As is shown in figure1, the whole sequence of our formaldehyde pathway is about 1500 base-pairs while the vector is 2000 base-pairs. SDS-PAGE analysis(figure 2) also shows 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 |
|
Figure3 illustrates the fluorescence intensity change induced by formaldehyde along with an interval of 2 hours. The peak value occurs after 6 hours, which means the detecting results can be seen with naked-eyes after only 6 hours. As is shown in figure 4, compared to the blank control, experimental group with formaldehyde induction turns to pink apparently, meaning the designed reporter pathway has 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) |
||
It is worth to be mentioned that the team OUC help us validate the result. | ||
Moreover, in order to set up the corresponding relationship between the quantity of formaldehyde and the fluorescence value, we prepared a series of concentrations of formaldehyde(figure5a.). We found out that from the concentration of 300 micromole to 600 micromole, a preferable equation of linear regression could be obtained(figure5b.), which laid the cornerstone for creating precise and sensitive detecting devices. |
||
a) |
||
b) |
||
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) |
||
Then we incubated our recombinant E. coli for more than 10 hours to test their reponse growth rates(figure 6a) and tolerance(figure 6b) to different concentrations of formaldehyde. |
||
Figure6a.Response growth curve for recombinant bioluminescent Escherichia coli BL21 to different concentration of formaldehyde | ||
Figur6b.The tolerance of recombinant bioluminescent Escherichia coli BL21 to various concentration of formaldehyde | ||
Finally, we tested the selectivity of our formaldehyde pathway by inducing the recombinant E. coli with acetaldehyde, DMSO and 6 other aldehydes. The results(figure 7) demonstrated good selsctivity of our recombinant plasmids. | ||
Figure7.Fluorescence test of various aldehydes using recombinant bioluminescent Escherichia coli BL21 |
We first analyzed the product by dual-enzyme digestion and electrophoresis(figure8). As can be seen, our hydrogen sulfide sensing sequence is over 3,000 base pairs. |
|
Figure8.Whole-cell sequence dual-enzyme digestion |
|
Next, we conducted a plate sensitive assay to measure the S2– tolerance of E. coli cells with constructed probe pathway. All plates were incubated at 37℃ for 18 h before reading. No significant influence appeared to the growth of E. coli at a concentration lower than 10mmol/L. |
|
Figure 9. Tolerance test |
|
Cells were then 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. |
|
Figure10 a)RFP responsiveness of the detector system. | |
Figure10 b) A visible photograph of a). |
|
Finally, we examined the plasmid's selectivity against SO42-, SO32- and 4 other chemical reagents(figure 11). |
|
Figure 11.Test of selectivity. |
|
|
First we succefully detected the protein expression by SDS-Page(figure 12a) analysis and Western blot(figure 12b) analysis. |
|
Figure12a. Coomassie Brilliant Blue R-250-stained SDS-Page analysis of recombinant E.coli expressing hoxABCJ-terminator-hoxp-gfp |
Fingure 12b. Western blot analysis of recombinant E.coli expressing his-hoxA |
Fluorescence intensity remains stationary when IPTG is added. Whereas, it increases in a low hydrogen atmosphere. When the amount of hydrogen goes to an even higher level, fluorescence intensity increases apparently, meaning the designed report pathway works as expected(figure 13). |
|
Figure 13. Influence of H2 concentration on fluorescence expression |