Difference between revisions of "Team:Paris Bettencourt/Project/VitaminB2"
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The chromosome integration plasmid pKV6 was first altered to allow the insertion of the four genes by Golden Gate Assembly. This new plasmid was called p15.01.<br> | The chromosome integration plasmid pKV6 was first altered to allow the insertion of the four genes by Golden Gate Assembly. This new plasmid was called p15.01.<br> | ||
Then, the four genes were successfully inserted in p15.01 and cloned in <i>E. coli</i>.<br> | Then, the four genes were successfully inserted in p15.01 and cloned in <i>E. coli</i>.<br> | ||
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<img width="350px" src="https://static.igem.org/mediawiki/2015/6/61/PkV6_Map.jpg"/><br> | <img width="350px" src="https://static.igem.org/mediawiki/2015/6/61/PkV6_Map.jpg"/><br> | ||
− | <b>Figure 1: </b><i>pKV6 is an integrative plasmid we got from Christian Solem for repetitive marker free chromosomal integration. </i> | + | <b>Figure 1: </b><i>pKV6 is an integrative plasmid we got from Christian Solem for repetitive marker free chromosomal integration. </i><br> |
− | + | <img width="350px" src="https://static.igem.org/mediawiki/2015/6/6b/P15.01_Map.jpg"/><br> | |
− | <b>Figure 2: </b><i>p15.01 is a derivative plasmid from pKV6, where a pair of BbsI restriction enzyme sites were introduced for Golden gate assembly. </i> | + | <b>Figure 2: </b><i>p15.01 is a derivative plasmid from pKV6, where a pair of BbsI restriction enzyme sites were introduced for Golden gate assembly. </i><br> |
<img width="350px" src="https://static.igem.org/mediawiki/2015/0/0c/P15_06_Map.jpg"/><br> | <img width="350px" src="https://static.igem.org/mediawiki/2015/0/0c/P15_06_Map.jpg"/><br> | ||
− | <b>Figure 3: </b><i>p15.06 is a derivative plasmid from p15.01 after golden gate assembly of Vitamin B2 pathway. Here RibA cassette has P48 synthetic promoter and all the other cassettes RibD, RibE, RibT have P25 synthetic promoter. </i> | + | <b>Figure 3: </b><i>p15.06 is a derivative plasmid from p15.01 after golden gate assembly of Vitamin B2 pathway. Here RibA cassette has P48 synthetic promoter and all the other cassettes RibD, RibE, RibT have P25 synthetic promoter. </i><br> |
<img width="350px" src="https://static.igem.org/mediawiki/2015/2/20/P15_07_Map.jpg"/><br> | <img width="350px" src="https://static.igem.org/mediawiki/2015/2/20/P15_07_Map.jpg"/><br> | ||
− | <b>Figure 4: </b><i>p15.07 is a derivative plasmid from p15.01 after golden gate assembly of Vitamin B2 pathway. Here RibA and RibT cassettes have P48 synthetic promoter and rest two cassettes RibD, RibE have P25 synthetic promoter.</i> | + | <b>Figure 4: </b><i>p15.07 is a derivative plasmid from p15.01 after golden gate assembly of Vitamin B2 pathway. Here RibA and RibT cassettes have P48 synthetic promoter and rest two cassettes RibD, RibE have P25 synthetic promoter.</i><br><br> |
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After cloning, cells began to release riboflavin in their surrounding media, suggesting that the expression system was functioning in E. coli as well. <br> | After cloning, cells began to release riboflavin in their surrounding media, suggesting that the expression system was functioning in E. coli as well. <br> | ||
Riboflavin concentration corresponding to the fluorescence (at 565nm) of the supernatant was estimated on a standard curve.<br> | Riboflavin concentration corresponding to the fluorescence (at 565nm) of the supernatant was estimated on a standard curve.<br> | ||
− | <img width=" | + | <img width="450px" src="https://static.igem.org/mediawiki/2015/6/6f/Riboflavin_standard_curve.png"><br> |
<b>Figure 5: </b><i>Standard curve of riboflavin fluorescence</i><br><br> | <b>Figure 5: </b><i>Standard curve of riboflavin fluorescence</i><br><br> | ||
From the standard curve, we derived a linear equation mapping riboflavin concentration to fluorescence (fluorescence = 16344*x + 418).<br> | From the standard curve, we derived a linear equation mapping riboflavin concentration to fluorescence (fluorescence = 16344*x + 418).<br> | ||
− | <img width=" | + | <img width="500px" src="https://static.igem.org/mediawiki/2015/8/80/PB_overproduction_riboflavin.png"><br> |
<b>Figure 6: </b><i>Over-production of riboflavin by E.coli </i><br><br> | <b>Figure 6: </b><i>Over-production of riboflavin by E.coli </i><br><br> | ||
<i>E. coli</i> strains over-produce riboflavin up to 6µg/mL of media.<br><br> | <i>E. coli</i> strains over-produce riboflavin up to 6µg/mL of media.<br><br> | ||
− | <img width=" | + | <img width="300px" src="https://static.igem.org/mediawiki/parts/d/d2/B2_plate_riboflavin_diffusing_m9.png"><br> |
− | <b>Figure 7: </b><i> | + | <b>Figure 7: </b><i>E. coli strain diffusing riboflavin in plate</i><br><br> |
+ | <img width="400px" src="https://static.igem.org/mediawiki/2015/9/92/PB_projetB2_pic7.jpg"><br> | ||
+ | <b>Figure 8: </b><i>Riboflavin revelation by fluorescence</i><br><br> | ||
− | We characterized the functioning of promoter p48 in <i>E. coli</i> and BioBrick it(<a href="http://parts.igem.org/Part:BBa_K1678004">BBa_K1678004</a>).<br> | + | We characterized the functioning of promoter p48 in <i>E. coli</i> and BioBrick it (<a href="http://parts.igem.org/Part:BBa_K1678004">BBa_K1678004</a>).<br> |
Activity of p48 was shown by the expression of the fluorescent protein mCerulean.<br> | Activity of p48 was shown by the expression of the fluorescent protein mCerulean.<br> | ||
− | <img width=" | + | <img width="400px" src="https://static.igem.org/mediawiki/parts/b/bb/BB_fluorescence_chart_BB_04.png"><br> |
− | <b>Figure | + | <b>Figure 9: </b><i>Fluorescence of mCerulean promoted by p48</i><br><br> |
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− | <div class="column- | + | <h1 class="date one" id="attribution">Attributions</h1> |
− | <h1 class="date one" id="attribution"> | + | |
This project was designed and accomplished by Barthelemy Caron and Shazzad Hossain Mukit in consultation with Jason Bland, Ihab Boulas, Jake Wintermute and Antoine Decrulle. Most of the strains (DH5alpha, Top10, NEB turbo, STABLE) were kindly provided by Inserm U1001. Plasmids pKV6, PKV7 and pLB65 were provided by Christian Solem and Jianming Liu of National Food Institute, DTU (Denmark). Plasmid pNZ5319 for L.plantarum chromosomal integration was provided by Michiel Kleerebezem and Ingrid van Alen of NIZO food research, The Netherlands. pL2 backbone and two inserts, pele1 and eYFP, for positive control of Golden Gate assembly were provided by Antoine Decrulle. Special thanks to all the people for their important advice, and all the Paris Bettencourt team members for making this adventure such an amazing experience. | This project was designed and accomplished by Barthelemy Caron and Shazzad Hossain Mukit in consultation with Jason Bland, Ihab Boulas, Jake Wintermute and Antoine Decrulle. Most of the strains (DH5alpha, Top10, NEB turbo, STABLE) were kindly provided by Inserm U1001. Plasmids pKV6, PKV7 and pLB65 were provided by Christian Solem and Jianming Liu of National Food Institute, DTU (Denmark). Plasmid pNZ5319 for L.plantarum chromosomal integration was provided by Michiel Kleerebezem and Ingrid van Alen of NIZO food research, The Netherlands. pL2 backbone and two inserts, pele1 and eYFP, for positive control of Golden Gate assembly were provided by Antoine Decrulle. Special thanks to all the people for their important advice, and all the Paris Bettencourt team members for making this adventure such an amazing experience. | ||
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Latest revision as of 23:45, 19 November 2015