Team:INSA-Lyon/Results
From 2014.igem.org
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- | <div align="justify"> | + | <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> |
- | + | <p>Firstly, a <b> calibration curve </b> of the formation Nickel and DMG complexes was established. </p> | |
- | Nickel(II) | + | |
- | < | + | <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/> |
- | Firstly, a <b> calibration | + | 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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<div align="center"> | <div align="center"> | ||
<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" | ||
- | width="600px" | + | 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" | ||
- | width="600px"/> | + | width="600px"/> |
</div> | </div> | ||
- | 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 | + | <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 <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> | + | |
+ | <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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<div align="center"> | <div align="center"> | ||
<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> | width="500px"/> </div> | ||
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- | Taken together, these results show that the | + | <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> | ||
<br/> </div> | <br/> </div> |
Revision as of 13:47, 17 October 2014