Team:INSA-Lyon/Results

From 2014.igem.org

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<b>Figure 1 : Engineered bacteria Percentage of adhesion</b><br/>
<b>Figure 1 : Engineered bacteria Percentage of adhesion</b><br/>
<p align="justify"><i>csgA-</i>knockout <i>E. coli</i> strain was transformed with BBa_CsgA-WT (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404006">BBa_K1404006</a>); BBa_CsgA-His1 (<a href="http://parts.igem.org/Part:BBa_K1404007">BBa_K1404007</a>); BBa_CsgA-His2 (<a href="http://parts.igem.org/Part:BBa_K1404008">BBa_K1404008</a>). The corresponding positive and negative controls are, respectively, Wild-type <i>E.coli</i> curli producing strain transformed with the empty vector and <i>csgA-</i>-knockout <i>E. coli</i> strain transformed with the empty vector. <br/>
<p align="justify"><i>csgA-</i>knockout <i>E. coli</i> strain was transformed with BBa_CsgA-WT (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404006">BBa_K1404006</a>); BBa_CsgA-His1 (<a href="http://parts.igem.org/Part:BBa_K1404007">BBa_K1404007</a>); BBa_CsgA-His2 (<a href="http://parts.igem.org/Part:BBa_K1404008">BBa_K1404008</a>). The corresponding positive and negative controls are, respectively, Wild-type <i>E.coli</i> curli producing strain transformed with the empty vector and <i>csgA-</i>-knockout <i>E. coli</i> strain transformed with the empty vector. <br/>
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Strains with our parts, the positive and negative controls were cultured in a 24-wells microplate in M63 Mannitol during 24H at 30°C. The supernatant was removed and the OD600 measured, then the bacteria forming the biofilm were resuspended and the OD600 was measured in order to estimate the number of cells (<a href="https://static.igem.org/mediawiki/2014/8/80/Adhesion_test_protocole.pdf" target="_blank">See protocol for details</a>). The percentage of adhesion was calculated as follows:  
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Strains with our parts, the positive and negative controls were cultured in a 24-well microplate in M63 Mannitol during 24H at 30°C. The supernatant was removed and the OD600 measured, then the bacteria forming the biofilm were resuspended and the OD600 was measured in order to estimate the number of cells (<a href="https://static.igem.org/mediawiki/2014/8/80/Adhesion_test_protocole.pdf" target="_blank">See protocol for details</a>). The percentage of adhesion was calculated as follows:  
(OD600 of  the biofilm)/ (OD600 of  the supernatant + OD600 of the biofilm) <br/>
(OD600 of  the biofilm)/ (OD600 of  the supernatant + OD600 of the biofilm) <br/>
Different uppercase letters displayed on the graph  indicate significant differences  between strains (Tukey’s test, p < 0.05) <br/>
Different uppercase letters displayed on the graph  indicate significant differences  between strains (Tukey’s test, p < 0.05) <br/>
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<h5>Confocal Laser Scanning Microscopy Analyses</h5>
<h5>Confocal Laser Scanning Microscopy Analyses</h5>
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<div align="justify"><p><br/>For the Confocal Laser Scanning Microscopy biofilm acquisitions, all the strains were cultivated in 96-wells microplate in M63 Mannitol during 16H at 30°C (<a href="https://static.igem.org/mediawiki/2014/7/7e/Culture_confocal_analyse.pdf" target="_blank">See Protocol for details</a>). See results in <b>Figure 4</b>.</p>
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<div align="justify"><p><br/>For the Confocal Laser Scanning Microscopy biofilm acquisitions, all the strains were cultivated in 96-well microplate in M63 Mannitol during 16H at 30°C (<a href="https://static.igem.org/mediawiki/2014/7/7e/Culture_confocal_analyse.pdf" target="_blank">See Protocol for details</a>). See results in <b>Figure 4</b>.</p>
<img src="https://static.igem.org/mediawiki/2014/7/7d/Figureglobaleetoile2.png"  
<img src="https://static.igem.org/mediawiki/2014/7/7d/Figureglobaleetoile2.png"  
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<p></br>As no strains carrying our parts show a significant difference with the positive control, our part’s insertion doesn’t modify the biofilm formation properties. <b>The His-Tag and His2-Tag engineered CsgA doesn’t disturb the curli formation.</b></p>
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<p></br>As no strain carrying our parts showed a significant difference with the positive control, our part’s insertion doesn’t modify the biofilm formation properties. <b>The His-Tag and His2-Tag engineered CsgA doesn’t disturb the curli formation.</b></p>
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<p>A second method has been used, more <b>quantitative</b> and more precise (but more expensive) : <b> ICP-MS </b>. (<a href="https://static.igem.org/mediawiki/2014/f/f7/Ni_chelation.pdf" target="_blank">See Protocol for more details</a>)<br/> The metal content of the bacterial pellets were assayed. The quantity of chelated nickel for each strain was compared to the quantity of curlis formed by each strain.</p>
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<p>A second method has been used, more <b>quantitative</b> and more precise (but more expensive) : <b> ICP-MS (Inductively coupled plasma mass spectrometry)</b>. (<a href="https://static.igem.org/mediawiki/2014/f/f7/Ni_chelation.pdf" target="_blank">See Protocol for more details</a>)<br/> The metal content of the bacterial pellets were assayed. The quantity of chelated nickel for each strain was compared to the quantity of curlis formed by each strain.</p>
<div align="center">
<div align="center">
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<p> Different lowercase letters displayed on the graph  indicate significant differences  between strains (Tukey’s test, p < 0.05). Error bars represent standard deviations.</p>
<p> Different lowercase letters displayed on the graph  indicate significant differences  between strains (Tukey’s test, p < 0.05). Error bars represent standard deviations.</p>
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<p>Taken together, these results show that the CsgA- Strain with part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404008">BBa_CsgA-His2</a> chelates twice as much as strain CsgA- with part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404007">BBa_CsgA-His1</a>. That means that <b>only two His-tags on C-term can improve the natural nickel chelation capacities of CsgA </b>. CsgA with a single His-tag did not perform better than a wild-type CsgA. It can be explained by the conformation of the His1-tag which could be folded on the side of CsgA, as presented in the <a href="https://2014.igem.org/Team:INSA-Lyon/Molecular#nichelation" target="_blank">modelisation section</a>. Potentially, further increase of the amount of His-tags could improve the nickel accumulation capacities of CsgA. </p>
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<p>Taken together, these results show that the CsgA- Strain with part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404008">BBa_CsgA-His2</a> chelates twice as much as strain CsgA- with part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404007">BBa_CsgA-His1</a>. That means that <b>only two His-tags on C-term can improve the natural nickel chelation capacities of CsgA </b>. CsgA with a single His-tag did not perform better than a wild-type CsgA. It can be explained by the conformation of the His1-tag which could be folded on the side of CsgA, as presented in the <a href="https://2014.igem.org/Team:INSA-Lyon/Molecular#nichelation" target="_blank">modelization section</a>. Potentially, further increase of the amount of His-tags could improve the nickel accumulation capacities of CsgA. </p>
<br/> </div>
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<div align="justify">
<div align="justify">
<p>
<p>
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To adress biosafety issues linked with GMOs, we worked on destroying our bacteria after letting them grow in a biofilm. As the captured metal is extracellular and Curli proteins are very resistant to environmental changes, alive bacteria are not needed for our biofilter. Our goal was to obtain a biomaterial made out of modified Curli able to chelate nickel.  
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To address biosafety issues linked with GMOs, we worked on destroying our bacteria after letting them grow in a biofilm. As the captured metal is extracellular and Curli proteins are very resistant to environmental changes, alive bacteria are not needed for our biofilter. Our goal was to obtain a biomaterial made out of modified Curli able to chelate nickel.  
</p> </br>
</p> </br>
<p>
<p>
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</br>
</br>
</table></br>
</table></br>
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<b>Figure 1 :Monitoring of bacteria kill after UV exposure</b> </div> <br/>
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<b>Figure 1: Monitoring of bacteria kill after UV exposure</b> </div> <br/>
No bacteria grew on LB plates after 15 minutes UV light exposure.</br>&rArr; <b>Bacterial growth can be stopped this way. </b></p></br>
No bacteria grew on LB plates after 15 minutes UV light exposure.</br>&rArr; <b>Bacterial growth can be stopped this way. </b></p></br>
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<tr>
<tr>
   <td><div align="center"><figcaption>
   <td><div align="center"><figcaption>
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<b>Figure 2 : PCR gel after UV exposure  </b>  </figcaption></div></td>
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<b>Figure 2: PCR gel after UV exposure  </b>  </figcaption></div></td>
</tr>
</tr>
</table> </div></br> Bacterial DNA seemed to be degraded after 10 min UV light exposure.</br>&rArr; <b>In consequence, UV light can be used to destroy DNA.</b> </p></br>
</table> </div></br> Bacterial DNA seemed to be degraded after 10 min UV light exposure.</br>&rArr; <b>In consequence, UV light can be used to destroy DNA.</b> </p></br>
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<p><div align="center"><i>Backlight</i></div></p></br>
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<p><div align="center"><i>Backlight</i></div></p>
<p><div align="center"><table>
<p><div align="center"><table>
<tr>
<tr>
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</table>
</table>
</br>
</br>
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<b>Figure 3 : Observations of bacteria exposed to UV and after backlight coloration : <br/>
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<b>Figure 3: Observations of bacteria exposed to UV and after backlight coloration : <br/>
alive bacteria appear in green and dead ones in red.</b> </div> <br/></div>
alive bacteria appear in green and dead ones in red.</b> </div> <br/></div>
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</table>
</table>
</br>
</br>
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<b>Figure 4 : Monitoring of bacteria heated at 60°C</b> </div></br>  
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<b>Figure 4: Monitoring of bacteria heated at 60°C</b> </div></br>  
Bacteria grew on LB plates even 45 min after being heated at 60°C. </br>&rArr; <b>60°C isn't enough high to kill bacteria.</b></p></br>
Bacteria grew on LB plates even 45 min after being heated at 60°C. </br>&rArr; <b>60°C isn't enough high to kill bacteria.</b></p></br>
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</tr>
</tr>
</table> </br>
</table> </br>
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<b>Figure 5 : Monitoring of bacteria heated at 70°C</b> </div></br>
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<b>Figure 5: Monitoring of bacteria heated at 70°C</b> </div></br>
No more bacteria grew on LB plates after 15min at 70°C<br/> &rArr;<b>Bacterial growth can be stopped as well as with UV light.</b></p></br>
No more bacteria grew on LB plates after 15min at 70°C<br/> &rArr;<b>Bacterial growth can be stopped as well as with UV light.</b></p></br>
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No DNA degradation at all.<br/>&rArr; <b>In consequence, unlike UV light,  temperature treatment doesn't destroy DNA.</b> </p><br/>
No DNA degradation at all.<br/>&rArr; <b>In consequence, unlike UV light,  temperature treatment doesn't destroy DNA.</b> </p><br/>
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<p><div align="center"><i>Backlight</i></div></p></br>
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<p><div align="center"><i>Backlight</i></div></p>
<p><div align="center"><table>
<p><div align="center"><table>
<tr>
<tr>
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</table>
</table>
</br>
</br>
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<b>Figure 7 : Observations of bacteria heated at 70°C and after backlight coloration</b>  
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<b>Figure 7: Observations of bacteria heated at 70°C and after backlight coloration</b>  
</div></br>  
</div></br>  
No difference of coloration was observed between the control and the samples heated at 70°C : indeed a lot of green-colored bacteria remained after 45 min of heating.<br/>&rArr; <b>Temperature isn’t enough to kill bacteria just like UV light.</b></p></br>
No difference of coloration was observed between the control and the samples heated at 70°C : indeed a lot of green-colored bacteria remained after 45 min of heating.<br/>&rArr; <b>Temperature isn’t enough to kill bacteria just like UV light.</b></p></br>
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<p><p align="justify">
<p><p align="justify">
As a follow-up to the exploration of curli production and nickel chelation, we want to know the kinetics behind the 70 base-pair long promoter sequence that we used during the whole summer.
As a follow-up to the exploration of curli production and nickel chelation, we want to know the kinetics behind the 70 base-pair long promoter sequence that we used during the whole summer.
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In fact, it has the interesting property of being activated at 37°C instead of the 30°C of the natural 750 base-pair CsgA promoter from where it is originally isolated. However, we explored these two promoters' kinetics at 30 and 37°C by inserting a GFP downstream.
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In fact, it has the interesting property of being activated at 37°C instead of the 30°C of the natural 750 base-pair <i>csgA</i> promoter from where it is originally isolated. However, we explored these two promoters' kinetics at 30 and 37°C by inserting a GFP downstream.
<div align="center">
<div align="center">
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<img src= "https://static.igem.org/mediawiki/parts/f/fb/2.png" alt="<b>Figure 1 : GFP expression plasmid for promoter characterization. The promoter sequence can either be the 70 (P70) or the 750 base-pair promoter sequence.</b>"/></br>
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<img src= "https://static.igem.org/mediawiki/parts/f/fb/2.png"></br>
</div>
</div>
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On a pKK backbone, two essential parts have been assembled: a promoter and a reporter gene. The reporter gene in this case is always the same: GFP. However, the promoter is different for each construction.
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<div align="center"><figcaption><b>Figure 1: GFP expression plasmid for promoter characterization. The promoter sequence can either be the 70 (P70) or the 750 base-pair promoter sequence.</b></figcaption>
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</div>
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</br>
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<p>On a pKK backbone, two essential parts have been assembled: a promoter and a reporter gene. The reporter gene in this case is always the same: GFP. However, the promoter is different for each construction.
On the one hand, P70 is the 70 base-pair long promoter sequence and when combined to the reporter GFP, the construction is called p70:GFP.
On the one hand, P70 is the 70 base-pair long promoter sequence and when combined to the reporter GFP, the construction is called p70:GFP.
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On the other hand, P750 is the 750 base-pair long promoter sequence coding for the inter-genic regulation region of curli production and combined to the reporter GFP, the construction is called P750:GFP.</br>
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On the other hand, P750 is the 750 base-pair long promoter sequence coding for the inter-genic regulation region of curli production and combined to the reporter GFP, the construction is called P750:GFP. These constructs allow to compare the expression of both promoters.
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The idea behind the two constructions is the correlate the GFP expression to the promoter activity.
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</p></p>
</p></p>
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<div align="center">
<div align="center">
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<img width="80%" src="https://static.igem.org/mediawiki/2014/5/58/PromoterExpression_EarlyStage.png" alt="<b>Figure 2 : Promoter expression as a function of the Optical Density. At early growth stage, P70 has a higher expression rate at 37°C relatively to the P750.</b>"></br>
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<img width="80%" src="https://static.igem.org/mediawiki/2014/5/58/PromoterExpression_EarlyStage.png"</br>
</div>
</div>
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<div align="center"><figcaption><b>Figure 2: Promoter expression as a function of the Optical Density. At early growth stage, P70 has a higher expression rate at 37°C relatively to the P750.</b></figcaption>
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</div>
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</br>
<p>
<p>
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<div align="center">
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<img width="80%" src="https://static.igem.org/mediawiki/2014/f/f9/PromoterExpression_LateStage.png" alt="<b>Figure 3 : Promoter expression as a function of the Optical Density. At mid/late growth stage, P750 has a delayed but higher expression rate at 30°C relatively to the P70.</b>">></br>
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<img width="80%" src="https://static.igem.org/mediawiki/2014/f/f9/PromoterExpression_LateStage.png"></br>
</div>
</div>
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<div align="center"><figcaption><b>Figure 3: Promoter expression as a function of the Optical Density. At mid/late growth stage, P750 has a delayed but higher expression rate at 30°C relatively to the P70.</b></figcaption></div>
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</br>
<p>
<p>

Latest revision as of 03:13, 18 October 2014

Curly'on - IGEM 2014 INSA-LYON

  • Curli characterization


  • Nickel chelation


  • Survival after UV and high temperature exposure


  • Promoter optimization and characterization