Team:INSA-Lyon/Results

From 2014.igem.org

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<div><p align="justify"><br/>Five complementary tests were performed to evaluate the ability of the modified cells to assemble functional curli: <b>1)</b> determination of the percentage of adherent cells to polystyrene in 24 wells-plates,<b> 2)</b> crystal violet staining of biofilm formed on polystyrene in 24 wells-plates, <b>3)</b> ability to bind the congo red,<b> 4)</b> biofilm maximum thickness measurement and biovolumes quantification of GFP-tagged biofilm observed with a confocal microscopy and <b> 5)</b> curli structure observation using Transmission Electron Microscopy (MET).</p>
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<div><p align="justify"><br/>Five complementary tests were performed to evaluate the ability of the modified cells to assemble functional curli:
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</br>
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<b>1)</b> determination of the percentage of adherent cells to polystyrene in 24 wells-plates,<b></br>
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2)</b> crystal violet staining of biofilm formed on polystyrene in 24 wells-plates, <b></br>
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3)</b> ability to bind the congo red,<b></br>
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4)</b> biofilm maximum thickness measurement and biovolumes quantification of GFP-tagged biofilm observed with a confocal microscopy and </br>
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<b> 5)</b> curli structure observation using Transmission Electron Microscopy (MET).</p>
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<div align=”center”><img src="https://static.igem.org/mediawiki/2014/0/0e/Adh%C3%A9rence.png" alt="Figure 1 : Engineered bacteria Percentage of adhesion"/></div>
<div align=”center”><img src="https://static.igem.org/mediawiki/2014/0/0e/Adh%C3%A9rence.png" alt="Figure 1 : Engineered bacteria Percentage of adhesion"/></div>
<b>Figure 1 : Engineered bacteria Percentage of adhesion</b><br/>
<b>Figure 1 : Engineered bacteria Percentage of adhesion</b><br/>
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<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 Wild-type <i>E.coli</i> curli producing strain transformed with with the empty vector and <i>csgA-</i>-knockout <i>E. coli</i> strain transformed with the empty vector respectively. <br/>
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<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 cultivated in 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 measured in order to estimate the number of cells (<a href="https://static.igem.org/mediawiki/2014/8/80/Adhesion_test_protocole.pdf">See protocol for details </a>). The percentage of adhesion was calculated as follow :  
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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/>
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Significant differences are indicated using uppercase letters, and different letters indicate significant differences (Tukey’s test, p < 0.05) <br/>
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Different uppercase letters displayed on the graph  indicate significant differences between strains (Tukey’s test, p < 0.05) <br/>
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These results show that <b>the percentage of adhesion is similar between the strains containing the three parts and the positive control, thus tagged CsgA were still functional</b>. CsgA with one or two tags from the P70 promoter were sufficient to form thick biofilms. </p><br/>
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These results show that <b>the percentage of adhesion is similar between the strains containing the three parts and the positive control, thus tagged CsgA were still functional</b>. CsgA with one or two tags expressed by the P70 promoter were sufficient to form thick biofilms. </p><br/>
<div align=”center”><img src=https://static.igem.org/mediawiki/2014/d/dc/Crystal_violet_2.png align=”center” alt="Figure 2 : Engineered bacteria Biofilm formation"/></div>
<div align=”center”><img src=https://static.igem.org/mediawiki/2014/d/dc/Crystal_violet_2.png align=”center” alt="Figure 2 : Engineered bacteria Biofilm formation"/></div>
<b>Figure 2 : Engineered bacteria Biofilm formation</b><br/>
<b>Figure 2 : Engineered bacteria Biofilm formation</b><br/>
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  <p align=" justify ">The cells were grown as described as figure 1. <br/>
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  <p align=" justify ">The cells were cultured as described in figure 1. <br/>
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<div align="justify"><p>The supernatant was removed and the remaining biofilm was fixed to the microplate by heat treatment at 80°C during 1H. The violet crystal solution was added in each well in order to stain the cells and the wells were washed with water to remove crystal violet in excess (<a href="https://static.igem.org/mediawiki/2014/e/ef/Crystal_Violet_protocole.pdf">See protocol for details </a>).<br/>
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<div align="justify"><p>The supernatant was removed and the remaining biofilm was fixed to the microplate by heat treatment at 80°C during 1H. The crystal violet solution was added in each well in order to stain the cells and the wells were washed with water to remove crystal violet in excess (<a href="https://static.igem.org/mediawiki/2014/e/ef/Crystal_Violet_protocole.pdf" target="_blank">See protocol for details</a>).<br/>
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Violet crystal staining shows that <b>the strain containing the three parts could form a biofilm like the positive control, thus tagged CsgA were still functional</b>. CsgA with one or two tags from the P70 promoter were sufficient to form thick biofilms.</p></div> </p>
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Crystal violet staining shows that <b>the strain containing the three parts could form a biofilm like the positive control. Thus tagged CsgA were still functional</b>. CsgA with one or two tags expressed by the P70 promoter were sufficient to form thick biofilms.</p></div> </p>
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<div align=”center”><img src=" https://static.igem.org/mediawiki/2014/8/81/Congo_Red_2.png" align=”center” alt="Figure 3 : Engineered bacteria curli production"/></div>
<div align=”center”><img src=" https://static.igem.org/mediawiki/2014/8/81/Congo_Red_2.png" align=”center” alt="Figure 3 : Engineered bacteria curli production"/></div>
<b>Figure 3 : Engineered bacteria curli production</b><br/>  
<b>Figure 3 : Engineered bacteria curli production</b><br/>  
<p align="justify">Strains are the same as in figure 1. <br/>
<p align="justify">Strains are the same as in figure 1. <br/>
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Strains with our parts, the positive and negative control were cultivated in M63 Mannitol at 30°C and 180rpm. After centrifugation, the supernatant was removed and the cell pellet was resuspended in the Congo Red solution, in order to specifically stain the curli. The samples were centrifuged again and the pellets observed (See protocol for more details). <br/>
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Strains with our parts, the positive and negative control were cultured in M63 Mannitol at 30°C and 180rpm. After centrifugation, the supernatant was removed and the cell pellet was resuspended in the Congo Red solution, in order to specifically stain the curli. The samples were centrifuged again and the pellets were observed (<a href="https://static.igem.org/mediawiki/2014/3/39/CongoRed.pdf" target="_blank">See protocol for more details</a>). <br/>
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Congo Red staining shows that <b>the CsgA with one or two tag from the P70 promoter allows to form curli fiber</b> which are able to bind congo red.<br/></p>
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Congo Red staining shows that <b>the CsgA with one or two tags expressed by the P70 promoter allows to form curli fibers</b> which are able to bind Congo Red.<br/></p>
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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">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><b>Figure 4: Engineered bacteria biofilm characterization and quantification using Confocal Laser Scanning Microscopy</p></b>
<p><b>Figure 4: Engineered bacteria biofilm characterization and quantification using Confocal Laser Scanning Microscopy</p></b>
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<p>All the strains used are constitutively fluorescent to allow detection with confocal laser microscopy (ZEISS LSM510 META, 40X/1.3OILDIC, laser Argon 4 lines 30 W 458 nm, 477 nm, 488 nm, 514 nm, <a href="https://static.igem.org/mediawiki/2014/7/7e/Culture_confocal_analyse.pdf">See Protocol</a>). Positive control/CsgA+ (Wild-type <i>E. coli</i> curli producing strain); Negative control/CsgA- (<i>csgA</i>-knockout <i>E. coli</i> strain); BBa_CsgA (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404006">BBa_K1404006</a>); BBa_CsgAHis1 (<a href="http://parts.igem.org/Part:BBa_K1404007">BBa_K1404007</a>); BBa_CsgAHis2 (<a href="http://parts.igem.org/Part:BBa_K1404008">BBa_K1404008</a>). <b>A)</b> Biofilm sections obtained by Z-stack acquisitions. <b>B)</b> Biofilm 3D reconstruction using IMARIS® from acquisitions in A). <b>C) </b>Bio-volume quantification and maximum of thickness measurement using COMSTAT2 (ImageJ). The strain marked with a star is significantly different from all others (Tukey’s test, p<0.05).</p>
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<p>All the strains used are constitutively fluorescent to allow detection with confocal laser microscopy (ZEISS LSM510 META, 40X/1.3OILDIC, laser Argon 4 lines 30 W 458 nm, 477 nm, 488 nm, 514 nm, <a href="https://static.igem.org/mediawiki/2014/7/7e/Culture_confocal_analyse.pdf" target="_blank">See Protocol</a>). Positive control/CsgA+ (Wild-type <i>E. coli</i> curli producing strain); Negative control/CsgA- (<i>csgA</i>-knockout <i>E. coli</i> strain); BBa_CsgA (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404006">BBa_K1404006</a>); BBa_CsgAHis1 (<a href="http://parts.igem.org/Part:BBa_K1404007">BBa_K1404007</a>); BBa_CsgAHis2 (<a href="http://parts.igem.org/Part:BBa_K1404008">BBa_K1404008</a>). <b>A)</b> Biofilm sections obtained by Z-stack acquisitions. <b>B)</b> Biofilm 3D reconstruction using IMARIS® from acquisitions in A). <b>C) </b>Bio-volume quantification and maximum of thickness measurement using COMSTAT2 (ImageJ). The strain marked with a star is significantly different from all others (Tukey’s test, p<0.05).</p>
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<p></br>As no strains carrying our parts show a significant difference with the positive control, our parts insertion doesn’t modify the biofilm formation property. <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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</h6>
</h6>
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<p>For the Transmission Electron Microscopy, all the strains were cultivated in conditions that allow the formation of the curli : 48h (for a 50 mL culture),  28⁰C temperature and low agitation. The ammonium molybdate was used as a negative colorant (Microscope: MET PHILIPS CM120). </p>
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<p>For the Transmission Electron Microscopy, all the strains were cultured in conditions that allow curli formation: 48h (for a 50 mL culture),  28⁰C temperature and low agitation. The ammonium molybdate was used as a negative colorant (Microscope: MET PHILIPS CM120). </p>
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<img src="https://static.igem.org/mediawiki/2014/8/8a/Figure5_MET.png"  
<img src="https://static.igem.org/mediawiki/2014/8/8a/Figure5_MET.png"  
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<h6> General Conclusion
<h6> General Conclusion
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<p><br/><b>The expression of CsgA derivatives</b> from the p70 <i>csg</i> promoter carried by the psb1c3 plasmid leads to <b>functional CsgA</b>, and allows <i>E. coli</i> to <b>stick and form biofilm</b>. Moreover, our results show that the <b>addition of one or two His-Tag on C-term of CsgA doesn’t disturb the normal properties of curli</b> (sturdiness, adhesion, structure and folding of CsgA).</p><br/></div></li>
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<p><br/><b>The expression of CsgA derivatives</b> expressed by the p70 <i>csg</i> promoter carried by the psb1c3 plasmid leads to <b>functional CsgA</b>, and allows <i>E. coli</i> to <b>stick and form biofilm</b>. Moreover, our results show that the <b>addition of one or two His-Tag on the C-term of CsgA doesn’t disturb the normal properties of curli</b> (sturdiness, adhesion, structure and folding of CsgA).</p><br/></div></li>
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<p>
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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_CsgA-WT (BBa_K1404006)</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404007">BBa_CsgA-His1 (BBa_K1404007)</a>,or <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404008">BBa_CsgA-His2 (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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<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 one of the constructions (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404006">BBa_CsgA-WT (BBa_K1404006)</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404007">BBa_CsgA-His1 (BBa_K1404007)</a>,or <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404008">BBa_CsgA-His2 (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>
<div align="center">
<div align="center">
<img src="https://static.igem.org/mediawiki/2014/9/9a/DMGexplain.png" alt="DMG explanation"  
<img src="https://static.igem.org/mediawiki/2014/9/9a/DMGexplain.png" alt="DMG explanation"  
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<b>Figure 1 : DMG-Ni complex formation with or without chelating bacteria</b><br/>
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<p>Firstly, a <b> calibration curve </b> of the formation Nickel and DMG complexes was established. </p>
<p>Firstly, a <b> calibration curve </b> of the formation Nickel and DMG complexes was established. </p>
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<p>Then, strains were assayed for biofilm nickel absorption on liquid cultures using the calibration curve, measuring the OD of the complex formed for each strain at 554nm.<br/>
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<p>Then, liquid cultured strains were assayed for biofilm nickel absorption using the calibration curve, after measuring the OD of the complex formed for each strain at 554nm. (<a href="https://static.igem.org/mediawiki/2014/0/01/Ni_chelation_DMG_n.pdf" target="_blank">See Protocol for details</a>)<br/>
Although quantification is possible, this technique lacks precision and is more suited for <b>qualitative</b> studies. However, it is a cheaper alternative to ICP-MS. </p>
Although quantification is possible, this technique lacks precision and is more suited for <b>qualitative</b> studies. However, it is a cheaper alternative to ICP-MS. </p>
<div align="center">
<div align="center">
<img src="https://static.igem.org/mediawiki/2014/e/ea/Wikiphotogamme.png" alt="photo de la gamme"  
<img src="https://static.igem.org/mediawiki/2014/e/ea/Wikiphotogamme.png" alt="photo de la gamme"  
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<img src="https://static.igem.org/mediawiki/2014/3/33/SetcalibrationDMG2.png" alt="gamme graphe"  
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<b>Figure 2 : Calibration set picture</b><br/>
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<img src="https://static.igem.org/mediawiki/2014/5/57/SetcalibrationDMG3.png" alt="gamme graphe"  
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<b>Figure 3 : Calibration set : DMG-Ni complex OD554 (optical density)=f([Ni(II)]</b><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">BBBa_CsgA-His2</a> is less colored than the others, which shows that only this part allows to capture more nickel.
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<p>Nickel-DMG complex colorimetry measurement follows a <b>linear regression</b> for concentrations  from 20uM to 100uM. Visually, we can see gradient from transparency (at [20uM]) to pink (at [100uM]). This visual method allows us to compare the Ni chelation between our strains. The paler 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">BBBa_CsgA-His2</a> is less colored than the others, which shows that this part allows to capture more nickel.
These results show that <b>the part BBa_CsgA-His2 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_CsgA-WT</a> and part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404007">BBa_CsgA-His1</a>. </p>
These results show that <b>the part BBa_CsgA-His2 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_CsgA-WT</a> and part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1404007">BBa_CsgA-His1</a>. </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>. 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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<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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<img src="https://static.igem.org/mediawiki/2014/b/b5/ICPresults.png" alt="ICP"  
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<img src="https://static.igem.org/mediawiki/2014/e/e8/ICPresults2.png" alt="ICP"  
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<b>Figure 4 : ICP-MS results : Ni(II) chelated for each construction linked to the quantity of curlis</b><br/> </div>  
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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> 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 more than 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. Potentially, further increasing 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>
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           </ul>
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     <li><a href="#contenu3" onclick="$('#contenu3').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Survival after UV and high temperature exposure</h1></a><hr/></li>
     <li><a href="#contenu3" onclick="$('#contenu3').slideToggle('slow')"><h1><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Survival after UV and high temperature exposure</h1></a><hr/></li>
           <ul id="contenu3" style="list-style-type: none !important;display:none;">
           <ul id="contenu3" style="list-style-type: none !important;display:none;">
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<li>
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<p>
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<div align="justify"><p>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, live 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/>
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<div align="justify">
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<p>To find the best way to degrade bacteria and DNA, the following protocol was used to test the influence of UV light and temperature separately : <br/>
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<p>
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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.  
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</p> </br>
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<p>
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In order to find the best way to degrade bacteria and DNA, the <a href="https://static.igem.org/mediawiki/2014/1/1e/UV_temperature.pdf" target="_blank">following protocol</a>  was used to test the influence of UV light and temperature separately : </br>
<ul>
<ul>
-
<li> Wells containing M63 cultures of strain 227 were put under UV light / at 60 or 70°C for different lengths of time. Well contents were then gradually transferred into Eppendorf  and diluted (100, 300, 900 and 2700 fold).<br/>  
+
<li> Wells containing M63 cultures of strain 227 were put under UV light or exposed to heat treatments (at 60 or 70°C) for different lengths of time. Well contents were then gradually transferred into Eppendorfs and diluted (100, 300, 900 and 2700 fold).</li>  
-
<li> LB plates (without antibiotic) corresponding to UV/temperature exposure times (+ one plate for control) were then spotted with s227 different concentrations in order to be able to count survival bacteria after incubation at 37°C.<br/>
+
<li> LB plates (without antibiotic) corresponding to UV/temperature exposure times (+ one plate for control) were then spotted with different concentrations of strain 227 in order to be able to count bacterial survival after incubation at 37°C.</li>
-
<li> Genomic DNA was extracted from s227 concentrated culture. From the solution obtained, Curli promoter(750 bp) was amplified by PCR with Q5 polymerase and designed primers. <br/>
+
<li> Genomic DNA was extracted from strain 227 concentrated culture. From the solution obtained, Curli promoter(750 bp) was amplified by PCR with Q5 polymerase and designed primers. </li>
-
<li>Epifluorescence observations were made after Back Light coloration with 200µL s227 liquid cultures.</p><br/> <br/>  
+
<li>Epifluorescence observations were made after Back Light coloration of 227 strain liquid cultures.</li>
</ul>
</ul>
 +
</p>
 +
</br>
 +
 +
<p><h6> UV light influence </h6></p><br/>
-
<p><h6><i> UV light influence </i></h6></p><br/>
+
<p><div align="center"><i>LB plates</i></div></p>
-
<p><table>
+
<p><div align="center"><table>
<tr>
<tr>
-
   <td><img src="https://static.igem.org/mediawiki/2014/e/ed/PlatesUV1.png" alt="control plate" height="150 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/e/ed/PlatesUV1.png" alt="control plate" height="200 px"/></td>
-
   <td><img src="https://static.igem.org/mediawiki/2014/5/58/PlatesUV2.png" alt="5 min" height="150 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/5/58/PlatesUV2.png" alt="5 min" height="200 px"/></td>
-
   <td><img src="https://static.igem.org/mediawiki/2014/5/54/PlatesUV3.png" alt="10min" height="150 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/5/54/PlatesUV3.png" alt="10min" height="200 px"/></td>
-
  <td><img src="https://static.igem.org/mediawiki/2014/6/6a/PlatesUV4.png" alt="15min" height="150 px"/></td>
+
-
  <td><img src="https://static.igem.org/mediawiki/2014/b/bb/PlatesUV5.png" alt="20 min" height="150 px"/></td>
+
</tr>
</tr>
<tr>
<tr>
Line 169: Line 185:
   <td><div align="center"><figcaption>5 min UV</figcaption></td></div>
   <td><div align="center"><figcaption>5 min UV</figcaption></td></div>
   <td><div align="center"><figcaption>10 min UV</figcaption></td></div>
   <td><div align="center"><figcaption>10 min UV</figcaption></td></div>
 +
</tr>
 +
</table></div>
 +
<div align="center">
 +
<table>
 +
<tr>
 +
  <td><img src="https://static.igem.org/mediawiki/2014/6/6a/PlatesUV4.png" alt="15min" height="200 px"/></td>
 +
  <td><img src="https://static.igem.org/mediawiki/2014/b/bb/PlatesUV5.png" alt="20 min" height="200 px"/></td>
 +
</tr>
 +
<tr>
   <td><div align="center"><figcaption>15 min UV</figcaption></td></div>
   <td><div align="center"><figcaption>15 min UV</figcaption></td></div>
   <td><div align="center"><figcaption>20 min UV</figcaption></td></div>
   <td><div align="center"><figcaption>20 min UV</figcaption></td></div>
-
</tr>
+
</br>
-
</table>
+
</table></br>
-
No bacteria grew on LB plate after 15 minutes UV light exposure.<br/><b>Bacterian growth can be stopped this way. </b></p><br/>
+
<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>
-
<p><table><br/>
+
<p><div align="center"><i> DNA extraction</i></div></p>
 +
<p><div align="center"><table>
<tr>
<tr>
-
   <td><div align="justify"><figcaption>gel PCR</figcaption></td></div>
+
   <td><img src="https://static.igem.org/mediawiki/2014/d/d8/Gel_PCR_UV.png" alt="15min" height="200 px"/></td>
</tr>
</tr>
-
</table> <br/> Bacterian 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/>
+
<tr>
 +
  <td><div align="center"><figcaption>
 +
<b>Figure 2: PCR gel after UV exposure  </b>  </figcaption></div></td>
 +
</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>
 +
 
-
<p><table>
+
<p><div align="center"><i>Backlight</i></div></p>
 +
<p><div align="center"><table>
<tr>
<tr>
-
   <td><img src="https://static.igem.org/mediawiki/2014/e/ee/BacklightControl_UV.png" alt="Control plate" width="200 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/e/ee/BacklightControl_UV.png " alt="Control plate" width="300 px"/></td>
-
   <td><img src="https://static.igem.org/mediawiki/2014/b/bc/BacklightUV15.png" alt="15 min" width="200 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/b/bc/BacklightUV15.png" alt="15 min" width="300 px"/></td>
-
   <td><img src="https://static.igem.org/mediawiki/2014/4/4b/BacklightUV20.png" alt="20min" width="200 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/4/4b/BacklightUV20.png" alt="20min" width="300 px"/></td>
</tr>
</tr>
<tr>
<tr>
-
   <td><div align="center"><figcaption>Control plate</figcaption></td></div>
+
   <td><div align="center"><figcaption>Control plate</figcaption></div></td>
-
   <td><div align="center"><figcaption>15 min UV</figcaption></td></div>
+
   <td><div align="center"><figcaption>15 min UV</figcaption></div></td>
-
   <td><div align="center"><figcaption>20 min UV</figcaption></td></div>
+
   <td><div align="center"><figcaption>20 min UV</figcaption></div></td>
</tr>
</tr>
-
<br/> Still some green-colored bacteria could be seen after 20 min UV exposure. <br/>  
+
</table>
-
&rArr;<b>UV light isn’t enough to kill bacteria.</b></p><br/><br/>
+
</br>
 +
<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>
 +
</br> Still some green-colored bacteria could be seen after 20 min UV exposure. </br>
 +
&rArr;<b>UV light isn’t enough to kill bacteria.</b></p></br></br>
-
<p><h6><i> Temperature influence </i></h6></p><br/>
 
-
<p><table>
+
<p><h6><b>Temperature influence</b></h6></p></br>
 +
 
 +
 
 +
<p><div align="center"><i>LB plates</i></div></p>
 +
<p><div align="center"><table>
<tr>
<tr>
-
   <td><img src=https://static.igem.org/mediawiki/2014/5/52/PlatesTemp60_1.png" alt="Control plate" width="200 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/5/52/PlatesTemp60_1.png" alt="Control plate" height="200 px"/></td>
-
   <td><img src="https://static.igem.org/mediawiki/2014/3/35/PlatesTemp60_2.png" alt="15 min" width="200 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/3/35/PlatesTemp60_2.png" alt="15 min" height="200 px"/></td>
-
  <td><img src="https://static.igem.org/mediawiki/2014/c/ce/PlatesTemp60_3.png" alt=30min" width="200 px"/></td>
+
-
  <td><img src="https://static.igem.org/mediawiki/2014/a/a2/PlatesTemp60_4.png" alt="45min" width="200 px"/></td>
+
</tr>
</tr>
<tr>
<tr>
-
   <td><div align="center"><figcaption>Control plate</figcaption></td></div>
+
   <td><div align="center"><figcaption>Control plate</figcaption></div></td>
-
  <td><div align="center"><figcaption>15 min 60°C</figcaption></td></div>
+
   <td><div align="center"><figcaption>15 min 60°C</figcaption></div></td>
-
   <td><div align="center"><figcaption>30 min 60°C</figcaption></td></div>
+
-
  <td><div align="center"><figcaption>45 min 60°C</figcaption></td></div>
+
</tr>
</tr>
-
</table><br/> Bacteria grew on LB plates even 45 min after being heated to 60°C. <br/>&rArr; <b>60°C isn't enough high to kill bacteria.</b></p><br/>
+
<tr>
 +
  <td><img src="https://static.igem.org/mediawiki/2014/c/ce/PlatesTemp60_3.png" alt=30min" height="200 px"/></td>
 +
  <td><img src="https://static.igem.org/mediawiki/2014/a/a2/PlatesTemp60_4.png" alt="45min" height="200 px"/></td>
 +
</tr>
 +
<tr>
 +
  <td><div align="center"><figcaption>30 min 60°C</figcaption></div></td>
 +
  <td><div align="center"><figcaption>45 min 60°C</figcaption></div></td>
 +
</tr>
 +
</table>
 +
</br>
 +
<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>
<p>So we tried experiments with a temperature of 70°C.</p> </br>
<p>So we tried experiments with a temperature of 70°C.</p> </br>
-
<p><table>
+
 
 +
 
 +
<p><div align="center"><i>LB plates</i></div></p>
 +
<p><div align="center"><table>
<tr>
<tr>
-
   <td><img src="" alt="15 min" width="200 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/2/2e/70_control.jpg" alt="Control plate" height="200 px"/></td>
-
   <td><img src="" alt="30min" width="200 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/1/19/70_15_min.jpg" alt="15 min" height="200 px"/></td>
-
   <td><img src="" alt="45min" width="200 px"/></td>
+
  </tr>
 +
<tr>
 +
   <td><div align="center"><figcaption>Control plate</figcaption></div></td>
 +
  <td><div align="center"><figcaption>15 min 70°C</figcaption></div></td>
</tr>
</tr>
<tr>
<tr>
-
   <td><div align="center"><figcaption>15 min 70°C</figcaption></td></div>
+
   <td><img src="https://static.igem.org/mediawiki/2014/a/a0/70_30_min.jpg" alt=30min" height="200 px"/></td>
-
   <td><div align="center"><figcaption>30 min 70°C</figcaption></td></div>
+
  <td><img src=https://static.igem.org/mediawiki/2014/d/d6/70_45_min.jpg alt=45min" height="200 px"/></td>
-
   <td><div align="center"><figcaption>45 min 70°C</figcaption></td></div>
+
  </tr>
 +
<tr>
 +
   <td><div align="center"><figcaption>30 min 70°C</figcaption></div></td>
 +
   <td><div align="center"><figcaption>45 min 70°C</figcaption></div></td>
</tr>
</tr>
-
</table><br/>No more bacteria on LB plate after 15min at 70°C<br/> &rArr;<b>Bacterian growth can be stopped as well as with UV light.</b></p><br/>
+
</table> </br>
 +
<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>
-
<p><table>
+
<p><div align="center"><i>DNA extraction</i></div></p>
 +
<p><div align="center"><table>
<tr>
<tr>
-
   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/3/3c/Gel_PCR_70.png" alt="control plate" width="200 px"/></td></div>
+
   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/3/3c/Gel_PCR_70.png" alt="PCR gel" width="200 px"/></div></td>
</tr>
</tr>
<tr>
<tr>
-
   <td><div align="center"><figcaption>PCR gel after DNA extraction from bacterian culture exposed to 70°C</figcaption></td></div>
+
   <td><div align="center"><figcaption><b>Figure 6 : PCR gel after DNA extraction from bacterial culture exposed to heat treatment with 70°C</b></figcaption></div></td>
</tr>
</tr>
-
</table> </br>No DNA degradation at all.<br/>&rArr; <b>In consequence, temperature doesn't enable to destroy DNA, in contrary to UV light.</b> </p><br/>
+
</table></div> </br>
 +
No DNA degradation at all.<br/>&rArr; <b>In consequence, unlike UV light,  temperature treatment doesn't destroy DNA.</b> </p><br/>
-
<p><table>
+
<p><div align="center"><i>Backlight</i></div></p>
 +
<p><div align="center"><table>
<tr>
<tr>
-
   <td><img src="https://static.igem.org/mediawiki/2014/d/df/BacklightControl_70.png" alt="control plate" width="200 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/d/df/BacklightControl_70.png" alt="control plate" width="300 px"/></td>
-
   <td><img src="https://static.igem.org/mediawiki/2014/9/9c/Backlight15_70.png" alt="15 min" width="200 px"/></td>
+
   <td><img src="https://static.igem.org/mediawiki/2014/9/9c/Backlight15_70.png" alt="15 min" width="300 px"/></td>
-
  <td><img src="https://static.igem.org/mediawiki/2014/f/f5/Backlight30_70.png" alt="30 min" width="200 px"/></td>
+
-
  <td><img src="https://static.igem.org/mediawiki/2014/2/24/Backlight45_70.png" alt="45 min" width="200 px"/></td>
+
</tr>
</tr>
<tr>
<tr>
-
   <td><div align="center">Control plate<figcaption></figcaption></td></div>
+
   <td><div align="center"><figcaption>Control plate</figcaption></div></td>
-
   <td><div align="center">15 min at 70°C<figcaption></figcaption></td></div>
+
   <td><div align="center"><figcaption>15 min at 70°C</figcaption></div></td>
-
  <td><div align="center">30 min at 70°C<figcaption></figcaption></td></div>
+
-
  <td><div align="center">45 min at 70°C<figcaption></figcaption></td></div>
+
</tr>
</tr>
-
</table></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/>
+
  <td><img src="https://static.igem.org/mediawiki/2014/f/f5/Backlight30_70.png" alt="30 min" width="300 px"/></td>
 +
  <td><img src="https://static.igem.org/mediawiki/2014/2/24/Backlight45_70.png" alt="45 min" width="300 px"/></td>
 +
</tr>
 +
<tr>
 +
  <td><div align="center"><figcaption>30 min at 70°C</figcaption></div></td>
 +
  <td><div align="center"><figcaption>45 min at 70°C</figcaption></div></td>
 +
</tr>
 +
</table>
 +
</br>
 +
<b>Figure 7: Observations of bacteria heated at 70°C and after backlight coloration</b>
 +
</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>
-
<p> To solve this last problem, bacteria were put in contact with ethanol absolute. The Back Light coloration gives the following picture. <br/> image benjamin</p><br/><br/>
+
<p> To solve this last problem, bacteria were put in contact with 70% ethanol. The Backlight coloration gives the following picture. </br>  
-
<p>These numerous experiments lead us to developp a protocol in three steps, illustrated by the drawing below :<br/>
+
<p><div align="center"><table>
-
<p><table>
+
<tr>
<tr>
-
   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/6/6b/Bilan.png" alt="schéma bilan" width="600 px" /></td></div>
+
   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/2/2a/Image_benjamin.png" alt="back light ethanol" width="300 px" /></div></td>
</tr>
</tr>
<tr>
<tr>
-
   <td><div align="center">Global strategy to kill bacteria<figcaption></figcaption></td></div>
+
   <td><div align="center"><b>Figure 8 : Backlight after DNA extraction of bacterial culture exposed to ethanol</b><figcaption></figcaption></div></td>
</tr>
</tr>
-
</table></br></p>  
+
</table></div></p></br>
 +
 
 +
<p>These numerous experiments lead us to develop a protocol in three steps, illustrated by the drawing below :</br>
 +
<div align="center"><table>
 +
<tr>
 +
  <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/6/6b/Bilan.png" alt="schéma bilan" width="600 px" /></div></td>
 +
</tr>
 +
<tr>
 +
  <td><div align="center"><figcaption><b>Figure 9 : Global strategy to kill bacteria</b></figcaption></div></td>
 +
</tr>
 +
</table>
 +
</div></p></br>  
</li>
</li>
           </ul>
           </ul>
Line 270: Line 348:
           <ul id="contenu4" style="list-style-type: none !important;display:none;">
           <ul id="contenu4" style="list-style-type: none !important;display:none;">
               <li>
               <li>
 +
<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.
 +
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.
-
<p align="justify"><p>
 
-
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 through this whole summer.
 
-
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 orginially isolated. However, we explored these two promoters' kinetics at 30 and 37°C by inserting a GFP downstream.
 
-
</p>
 
-
 
-
<p>
 
-
On a pKK backbone, two essential parts have been assembled: a promoter and a reporter. The reporter in this case is always the same: GFP. However, the promoter is different for each construction.
 
-
One a first hand, P70 is the 70 base-pair long promoter sequence and when combined to the reporter GFP is called p70:GFP.
 
-
On a second hand, P750 is the 750 base-pair long promoter sequence coding for the inter-genic regulation for curli production and combined to the report GFP is called P750:GFP.</br>
 
-
The idea behind the two constructions is shown in the figure below.
 
-
</p></p>
 
<div align="center">
<div align="center">
<img src= "https://static.igem.org/mediawiki/parts/f/fb/2.png"></br>
<img src= "https://static.igem.org/mediawiki/parts/f/fb/2.png"></br>
</div>
</div>
 +
 +
<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>
 +
</div>
 +
</br>
 +
 +
<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 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.
 +
 +
</p></p>
-
<p>
 
-
<b>Early growth stage promoter kinetics</b></br>
 
-
During the early growth stages at 37°C, we can observe that the P70 (orange) has a higher GFP expression level compared to the P750 (red).
 
-
However at 30°C, both P70 (light blue) and P750 (dark blue) have low GFP expression levels.
 
-
We conclude that P70 has the ability to prematurely activate downstream expression at 37°C
 
-
</p>
 
<div align="center">
<div align="center">
-
<img width="80%" src="https://static.igem.org/mediawiki/2014/5/58/PromoterExpression_EarlyStage.png"></br>
+
<img width="80%" src="https://static.igem.org/mediawiki/2014/5/58/PromoterExpression_EarlyStage.png"</br>
</div>
</div>
 +
<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>
 +
</div>
 +
</br>
<p>
<p>
-
<b>Late growth stage promoter kinetics</b></br>
+
<b>Early growth stage promoter kinetics</b></br>
-
<p align="justify">During the late growth stages at 37°C, we can observe that both P70 (orange) and P750 (red) have a downregulated GFP expression and are moving toward lower expressions.
+
<p align="justify">During the early growth stages at 37°C, we can observe that the P70 (orange) has a higher expression level compared to the P750 (red).
-
However at 30°C, P70 (light blue) stabilizes within the range of low GFP expressions and P750 (dark blue) reaches the highest GFP expression values.
+
However at 30°C, both P70 (light blue) and P750 (dark blue) have low expression levels.
-
We conclude that P750 has a delayed, albeit extremely high high-leveled, GFP expression at 30°C.
+
We conclude that P70 has the ability to prematurely activate downstream expression at 37°C.
</p></p>
</p></p>
 +
 +
<div align="center">
<div align="center">
<img width="80%" src="https://static.igem.org/mediawiki/2014/f/f9/PromoterExpression_LateStage.png"></br>
<img width="80%" src="https://static.igem.org/mediawiki/2014/f/f9/PromoterExpression_LateStage.png"></br>
</div>
</div>
 +
 +
<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>
 +
</br>
 +
 +
<p>
 +
<b>Mid/late growth stage promoter kinetics</b></br>
 +
<p align="justify">During the mid/late growth stages at 37°C, we can observe that both P70 (orange) and P750 (red) have a decreased promoter expression and are moving towards lower rates.
 +
However at 30°C, P70 (light blue) stabilizes within the range of low expression levels and P750 (dark blue) reaches the highest promoter expression rates.
 +
We conclude that P750 has a delayed, albeit extremely high-leveled, promoter expression at 30°C.
 +
</p></p>
 +
</ul>
</ul>

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