Team:INSA-Lyon/Results

From 2014.igem.org

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<div align="justify">What about chelation ? <br/>
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<div align="justify"><p> Nickel(II) chelation was evaluated in a CsgA- MG1655 background (in order to have only our modified or unmodified curlis at the surface of the strain) for each of the constructions (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404006">BBa_K1404006</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404007">BBa_K1404007</a>,or <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404008">BBa_K1404008</a>). Dimethylglyoxime (DMG) was used as a complexing reagent, which forms a pink-colored complex (peak absorption at 554nm) in the presence of Ni(II). </p>
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<p>Firstly, a <b> calibration curve </b> of the formation Nickel and DMG complexes was established. </p>
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Nickel(II) chelated for each of the constructions (Strain CsgA- with part BBa_K1404006, Strain CsgA- with part BBa_K1404007 and Strain CsgA- with part BBa_K1404008) is evaluated by using dimethylglyoxime (DMG) as the precipitating reagent. This is achieved by using absorbing properties of DMG-Ni(II) pink-colored complex (peak absorption at 554nm). <br/>
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<p>Then, strains were assayed for biofilm nickel absorption on liquid cultures using the calibration curve, b measuring the OD of the complex formed for each strain at 554nm.<br/>
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Firstly, a <b> calibration set </b> of Nickel and DMG was done. <br/>
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Although quantification is possible, this technique lacks precision and is more suited for <b>qualitative</b> studies. However, it is a cheaper alternative to mass spectrometry. </p>
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Then, strains were assayed for biofilm nickel absorption on liquid cultures using the calibration set, after measuring the OD of the complex formed for each strain at 554nm.<br/>
 
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This technique is more <b>qualitative</b> than quantitative due to the spectrometer precision. <br/>
 
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Here are the outcomes. </p><br/>
 
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<img src="https://static.igem.org/mediawiki/2013/e/ea/Wikiphotogamme.png" alt="photo de la gamme"  
<img src="https://static.igem.org/mediawiki/2013/e/ea/Wikiphotogamme.png" alt="photo de la gamme"  
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width="600px"/>  
<img src="https://static.igem.org/mediawiki/2014/6/63/SetcalibrationDMG.png" alt="gamme graphe"  
<img src="https://static.igem.org/mediawiki/2014/6/63/SetcalibrationDMG.png" alt="gamme graphe"  
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width="600px"/>  <br/> <br/>
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width="600px"/>   
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Nickel-DMG complex colorimetry measurement follows a <b>linear regression</b> from a concentration of 20uM to 100uM, linked to the gradient from transparency (at [20uM]) to pink (at [100uM]). This visual method allows us to compare the Ni chelation between our strains. The more the color is pale, the more Ni has been chelated. That’s why,  the complex formed with bacteria from strain CsgA- with part BBa_K1404008 is less colored than the others, chelating bacteria remained in the pellet with nickel fixed to their curlis.<br/>
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<p>Nickel-DMG complex colorimetry measurement follows a <b>linear regression</b> from a concentration of 20uM to 100uM, linked to the gradient from transparency (at [20uM]) to pink (at [100uM]). This visual method allows us to compare the Ni chelation between our strains. The more pale the color is, the more Ni has been chelated. The culture supernatant of CsgA- bacteria from strain with the part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404008">BBa_K1404008</a> is less colored than the others, which shows that only this part allows to capture more nickel.
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These results show that <b>the part BBa_K1404008 confers increased chelation</b> to strain CsgA-. It is shown that it chelates more than part BBa_K1404006 and part BBa_K1404007. <br/>
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These results show that <b>the part BBa_K1404008 confers increased chelation</b> to strain CsgA-. It is shown that it chelates more than part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404006">BBa_K1404006</a> and part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404007">BBa_K1404007</a>. </p>
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A second method has been used, more <b>quantitative</b> and more precise: <b> mass spectrometry </b>assays have been realized. The quantity of chelated nickel for each strain has been compared to the quantity of curlis formed by each strain.
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<p>A second method has been used, more <b>quantitative</b> and more precise (but more expensive) : <b> mass spectrometry </b>. The metal content of the bacterial pellets were assayed. The quantity of chelated nickel for each strain has been compared to the quantity of curlis formed by each strain.</p>
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Here are the results : <br/>
 
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<img src="https://static.igem.org/mediawiki/2014/1/10/ICP.png" alt="ICP"  
<img src="https://static.igem.org/mediawiki/2014/1/10/ICP.png" alt="ICP"  
width="500px"/> </div>
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Taken together, these results show that the constructed Strain CsgA- with part BBa_K1404008 chelates twice more than strain CsgA- with part BBa_K1404007. That means that <b>two Histags on C-term are twice more effective </b> than one. Moreover, one histag allows a better chelation than none, but not a really significative one. Significant differences are indicated using lowercase letters, and different letters indicate significant differences (Tukey’s test, p < 0.05). Error bars represent standard deviations.  
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<p>Significant differences are indicated using lowercase letters, and different letters indicate significant differences (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_K1404008</a>8 chelates twice more than strain CsgA- with part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404007">BBa_K1404007</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. Potentially, further increasing the amount of His-tags could improve the nickel accumulation capacities of CsgA. </p>
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Revision as of 13:47, 17 October 2014

Curly'on - IGEM 2014 INSA-LYON

  • Curli characterization


  • Nickel chelation


  • Survival after UV and high temperature exposure


  • Promoter optimization and characterization