http://2013.igem.org/wiki/index.php?title=Special:Contributions/Yzegman&feed=atom&limit=50&target=Yzegman&year=&month=2013.igem.org - User contributions [en]2024-03-28T23:49:22ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Paris_Bettencourt/Project/TargetTeam:Paris Bettencourt/Project/Target2013-10-29T03:50:24Z<p>Yzegman: </p>
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<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections</p><br />
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<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li><br />
<li>Identified a potential anti-TB activity of Pyridoxine at high doses.</li><br />
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<p></p><br />
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<div class="biocriks"><br />
<h2>BioBricks</h2><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li> <br />
<li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li><br />
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<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria</p><br />
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<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
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<a href="#Model"><br />
<div class="hlink"><br />
<h2>Skip to Modeling</h2><br />
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</a><br />
<a href="#Design"><br />
<div class="hlink"><br />
<h2>Skip to Design</h2><br />
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<a href="#Results"> <br />
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<h2>Skip to Results</h2><br />
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<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SirA is essential for <i>M. tuberculosis</i> persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced <a href="#Reference">(Pinto <i>et al</i> 2007)</a>. Currently, there are no drug candidates that are known to specifically inhibit SirA and conventional drug screens involve do not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. We designed a working drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA. To this end we cloned Ito <i>E. coli </i><span style="font-style: normal;">the sulfite reduction pathway</span> of <i>M. smegmatis</i>, a non-pathogenic mycobacterial relative of <i>M. Tuberculosis</i>. Our model overcomes the problem of long doubling time of <i>M. tuberculosis</i>. Specific inhibition of the sulfite reduction pathway is scored by comparing a drug screen of our <i>E. coli</i> construct <i>vs.</i> wild-type. Any drug candidates that have activity against both the wild-type <i>E. coli</i> and our construct are non-specific inhibitors of <i>E. coli</i> growth. However, any drug candidates that inhibit only the growth of our <i>E. coli </i>construct will be <span style="font-style: normal;">SirA</span><i> </i><span style="font-style: normal;">pathway specific.</span> <br />
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<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" width="535px"/></a><br />
<p><b>Figure 1: Overview of Targeted Drug Screen Design</b></p><br />
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<div id="Model"></div><br />
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<h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We used an <i>E. coli</i> model (iJR904) obtained from the <a href="http://bigg.ucsd.edu/bigg/main.pl">BiGG database</a> as a starting model to obtain wild-type growth rate (f = 0.9129 divisions/hour). We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f= -8e-13=0 divisions/hour indicating that the sulphite reduction pathway is essential for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth with the mycobacteria pathway reverts the growth phenotype back to wild-type levels (f = 0.9105 divisions/hour). We then wanted to expand our model to find new pathways that we could utilize for a targeted drug screen approach. We wrote a matlab script that finds all the essential reactions in <i>M. tuberculosis</i> and all the essential reactions in <i>E. coli</i>, and then tries to complement the essential reactions in the <i>E. coli</i> model with the essential reactions from <i>M. tuberculosis</i>. The model identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 metabolic reactions</a> that we could target. Additionally, due to the modular nature of the model, it can be used to find target-able metabolic reactions in any SBML file. The Matlab scripts can be found <a href="https://2013.igem.org/File:TargetFBA.zip">here</a> and requires <a href="http://opencobra.sourceforge.net/openCOBRA/Welcome.html">Cobra Toolbox 2.0</a> to function. Please visit the FBA page for a detailed list of <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">results</a>.<br />
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<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" width="267.5px"/></a></center><br />
<p><b>Figure 2: Biomass Flux through <i>E. coli</i> and mycoSIR E. coli</b><div style="font-size: 90%">Flux balance analysis was run using Cobra Toolbox 2.0 on <i>E. coli</i> sbml model iJR904 with and without SULR reaction. Additionally an <i>E. coli</i> sbml model was built with the SULR reaction replaced with a reaction representing the mycobacterial SirA reaction and FprA reaction, as well as ferredoxin FdxA as an additional species. The Biomass flux is restored to 99.75% of the wild-type level with the synthetic mycobacterial system.</div></p><br />
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<div id="Model"></div><br />
<h2>Structural Analysis of SirA</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Superimposing the structures of <i>M.tuberculosis</i> SirA and <i>E.coli </i> CysI reveals high homology, in particular of the active sites. Both proteins have the same symmetry (psuedo 2 fold) indicative of a common evolutionary origin. Our analysis highlighted important conserved residues, involved in substrate binding to be Arg97, Arg130, Arg166, Lys207. These positively charged residues are conserved in the sulphite/nitrite reductase family. In addition, 4 Cys residues are conserved for iron-sulphur binding. </p><br />
<p>The most profound structural differences between the two enzymes are found in the ferredoxin binding site and SirA's most C terminal residues and several surface loop regions due to deletions or insertions. A stark difference is a covalent bond formed between Cys161 (thiolate) and Tyr69 (C carbon atom) found adjacent to the redox center (Cu ions) in SirA. The covalently bound residues act as a secondary cofactor in tyrosyl radical stabilization. </p> <br />
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<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" width="267.5px"/></a></center><br />
<p><b>Figure 3: The superimposed 3D protein structures of SirA and CysI.</b><div style="font-size: 90%"> 303 amino acids are involved in superimposition with an rsmd of 1.41Å. All domains and loops of CysI are coloured purple, whilst SirA is coloured according to structural similarity with CysI: Red indicates poor alignment whilst blue indicates good alignment.</div></p><br />
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<div id="Model"></div><br />
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<h2>Identification of potential drug target binding sites</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Our structural analysis provided the basis for our drug target prediction. Using Chembl and swiss pdb, we have shown a predicted drug target site. Our calculation gives strong favour for a drug to be effective at this site. The calculation reflects the suitability of small molecules to the binding site under the Lipinski's Rule of 5.</p><br />
<p>The drug target is located at the interface of the three domains. This binding pocket exhibits a dense hydrophobic region. Our analysis targets 48 amino acids of SirA within 6Å of a modelled small drug molecule. Of these residues, only 6 amino acids are charged: His409, Asp453, Asp474, His500, Asp504 and Arg541.<br />
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<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" width="267.5px"/></a></center><br />
<p><b>Figure 4 Drug target locations in SirA </b><div style="font-size: 90%">A domain located in SirA, identified as a drug target through Chembl analysis.</div></p><br />
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<h2>Structure based pharmacophore modelling of mycobacterial Fpra</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Using LigandScount 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Fpra. Our search revealed Riboflavin (Vitamin B2) and Pyridoxine to be drug targets for Fpra. We used NADP interacting with the active site as the model of the pharmacore. Results showed pyridoxin to be a competitive inhibitor to NADP. Pyridoxin is a synthetic compound currently available as a prescribed drug. </p><br />
<p>Chembl analysis of Pyridoxine (vitamin B6) show that it's properties fulfill Lipinski's criteria of being an orally active drug in humans. These properties state that any small drug molecule must have: no more than 5 H bond donors, no more 10 H bond acceptors (N or O atoms), mol mass of less than 500 dalts and octanol-water partition coefficient log P of no greater than 5).</p> <br />
<p>We have shown the proposed properties of Pyridoxine's interaction with Fpra as a competitive inhibitor to NADP at Fpra's active site. The key amino acids at the active site are Ala205, GLN204 and Thr208. GLN204 and Ala205 act as hydrogen bond acceptors whilst Thr208 interacts with a H via van der waals forces. Pyridoxin is a smaller, more lipid soluble molecule than NADP, thus more fitting to Lipinski's criteria. </p> <br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" width="100%"/></a></center><br />
<p><b>Figure 5: </b><div style="font-size: 90%">Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div></p><br />
<center><a href="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" width="100%"/></a></center><br />
<p><b>Figure 6:</b></b><div style="font-size: 90%"> The interaction of Pyridoxine to its active site residues.</div> </p><br />
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<div id="Design"></div><br />
<h2>Synthetic Mycobacteria Pathway</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
We designed a synthetic <i>M.smegmatis-</i> derived sulfite reduction pathway containing sirA - the sulfite reductase, and two supporting genes that are required for its function in <i>E.coli</i>: fdxA and fprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The genes' sequences were taken from previous work describing their expression <a href="#Reference">(Pinto <i>et al</i> 2007)</a> in <i>E.coli</i> for purification and in vitro characterization; we removed restriction sites and codon optimized for expression in <i>E. coli</i>. The genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genesand were transformed into our knock-out mutant strains of <i>E. coli</i>. Data on Growth curves can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Notebook#target_Monday_30th_September.html">here</a>. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" width="535px"/></a><br />
<p><b>Figure 7: Growth curves of <i>E. coli</i> mycoSIR</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MycoSIR <i>E. coli</i> (red). No growth was detected for uninduced MycoSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
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<h2>Creation of Knock out Mutants</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We prepared two strains of <i>E. coli</i> which have the sulfite reduction pathway deleted: BL21 (DE3) <i>ΔCysI Δfpr ΔydbK</i> and BL21 (AI) <i>ΔCysI</i>. CysI is responsible for sulfite reduction in <i>E. coli</i>, while <i>fpr and ydbK</i> are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to<i> E. coli</i> in which it is NADPH dependant. These genes were also removed to ensure that they do not interfere with our system. <br />
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<div class="rightparagraph"><br />
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<h2>Synthetic Corn Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group <a href="#Reference">(2011 Barstow et al)</a>. In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" width="535px"/></a><br />
<p><b>Figure 8:</b> Growth curves of <i>E. coli</i> maizeSIR<div style="font-size: 90%"> BL21 (DE3) ΔcysI containing the MaizeSIR pathway (MaizeSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MaizeSIR <i>E. coli</i> (red). No growth was detected for uninduced MaizeSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
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<div id="Results"></div><br />
<h2>Results</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Upon successful cloning of the three genes into our <i>E. coli</i> deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in <i>M. tuberculosis</i> and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in <i>E. coli</i>.<br />
</p><br />
</br><br />
<p><b>Figure 9:</b> Growth of zmSIR <i>E. coli</i> on minimal media. <div style="font-size: 90%">BL21 (DE3) ΔcysI cells transformed with 1, 2 and 3 genes of the 3-gene zmSIR synthetic pathway were grown for 24 hours on minimal media supplemented with 25 uM IPTG (see methods), along with a WT BL21 (DE3) serving as a negative control, and an untransformed BL21 (DE3) ΔcysI, as negative control. Rescue of growth required all genes of the synthetic pathway (SIR, FNR and FD). </div></p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" width="535px"/></a><br />
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<h2>Z-score</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
The Z-score is a statistical measurement aimed at assessing the "hit effect" in a drug screen high throughput screening. It is a commonly used measurement that shows how well did the drug effect the growth of the assay strain and how significant is the decrease in growth.</p><br />
<p><br />
To calculate the Z-score we used our experimental <i>E. coli</i> strain BL21 (AI) ΔcysI that carries all three genes of the synthetic pathway (sirA, fprA, fdxA). We grew it in the M9 minimal media supplemented with amino acid sulfur dropout powder, in a 96 well plate. Four of the wells were "spiked" with antibiotics (Amp, Gent, Kan, and Spect). </p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
This served as a simulation of the drug screen without the actual drug library. Only the drug screen controls are used: growth in M9 as a negative control (no drugs) and growth in M9 + antibiotics as a positive control (a sure hit). We then compared the distribution of the growth (OD) in the negative control with the distribution of growth (OD) in the positive control. The Z-score shows the distance of the negative control mean from the positive control mean in negative control standard deviation units.</p><br />
<p><b>Our Z-score is: -10.2.</b></p><br />
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<br />
<h2>Z-factor</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is a measurement complementary to the Z-score. It measures the assay's quality based on the same data extracted from the same experiment made for the Z-score. This calculation gives an estimation of how far the negative controls are from the positive controls. It is a comparison of the two distributions which assumes that both distributions are normal and calculate how far 99% of the data points of each distribution are from each other.</p><br />
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</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is given on a scale from 0 to 1. Scores between 0.5 and 1 show that the assay is good and will enable testing in High throughput screens.</p><br />
<p><b>Our Z-factor score is 0.58.</b></p><br />
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<div style="clear: both;"></div><br />
<br />
<h2> MycoSir growth assays reveal the potential anti TB activity of Pyridoxine </h2><br />
<br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;Motivated by the results of our computational analysis, we attemped to use MycoSIR E. coli to assay the activity of pyridoxine, our candidate FprA inhibitor and a potential anti-TB compound. Briefly, we added both pyridoxine and a control compound, riboflavin, to growing cultures of both WT E. coli and MycoSIR E. coli. In all cases, cells were grown in minimal media, where our previous work demonstrates that the MycoSIR pathway is essential for viability.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;Our growth assays indicate that pyridoxine, at high doses, specifically inhibits the growth of MycoSIR E. coli and therefore acts specifically on the mycobacterial sulfur pathway. While the observed affinity is low, it could in principle be expanded through derivitivizaion and further screening.<br />
These results indicate that our MycoSIR E. coli are a practical tool for measuring drug activities. We have ordered several small drug libraries to assay with our strain, and we look forward to finding more candidate anti-TB drugs!</p><br />
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<div style="clear: both;"></div><br />
<br />
<div class="leftparagraph"> <br />
<center><a href="https://static.igem.org/mediawiki/2013/d/d5/PB_RiboflavinData.png"><img width="80%" src="https://static.igem.org/mediawiki/2013/d/d5/PB_RiboflavinData.png"/></a></center><br />
<p><b>Figure 10:</b>Riboflavin has no effect on the growth of WT or synthetic MycoSIR <i>E. coli.</i><div style="font-size: 90%"></div> The indicated quantities of riboflavin were dissolved in water and added to cultures of WT or MycoSIR E. coli in 4 biological replicates. No significant growth effects were observed. </p><br />
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</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/8/86/PyridoxineData.png"><img width="80%" src="https://static.igem.org/mediawiki/2013/8/86/PyridoxineData.png"></a></center><br />
<p><b>Figure 11:</b> MycoSIR E. coli growth assays reveal a potential anti-TB acitivity of pyridoxine at high doses. <div style="font-size: 90%"> The indicated quantities of pyridoxine were dissolved in water and added to cultures of WT or MycoSIR E. coli in 4 biological replicates. Both strains were grown in defined minimal media, where MycoSIR E. coli require our synthetic pathway for growth. Low pyridoxine doses had no detectable effects. However, a very high dose of pyridoxine (10 mg/mL) substantially inhibited the growth of MycoSIR E. coli yet showed no effect on WT growth. This suggests pyridoxine specifically inhibits the activity of the Mycobacterial SirA pathway. Derivativization or other methods could be used to further enhance the affinity and specificity of this compound.</div> </p><br />
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<div id="Reference"></div><br />
<h2>Literature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Global Alliance for TB Drug Development, Tuberculosis. Scientific blueprint for tuberculosis drug development, Tuberculosis (Edinb) 81 Suppl 1, 1–52 (2001).</li><br />
<br />
<li>World Health Organization, Global Tuberculosis Report 2012 (2012).</li><br />
<br />
<li>K. Raman, K. Yeturu, N. Chandra, targetTB: A target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis, BMC Syst Biol 2, 109 (2008).</li><br />
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<li>R. Pinto, J. S. Harrison, T. Hsu, W. R. Jacobs, T. S. Leyh, Sulfite Reduction in Mycobacteria, Journal of Bacteriology 189, 6714–6722 (2007).</li><br />
<br />
<li>B. Barstow C. M. Agapakis, P. M. Boyle, G. Grandl, P. A. Silver, E. H. Wintermute, A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism, J Biol Eng 5, 7 (2011).</li><br />
</ul><br />
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</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ. 2011 Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6:1290-1307.</li><br />
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<li>Schellenberger, J., Park, J. O., Conrad, T. C., and Palsson, B. Ø., BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions, BMC Bioinformatics, 11:213, (2010).</li><br />
<br />
<li>S. G. Franzblau et al., Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis, Tuberculosis 92, 453–488 (2012).</li><br />
<br />
<li>D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Drugs for bad bugs: confronting the challenges of antibacterial discovery, Nat Rev Drug Discov 6, 29–40 (2006).</li><br />
<br />
<li>M. Nakayama, T. Akashi, T. Hase, Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin, J. Inorg. Biochem. 82, 27–32 (2000).</li><br />
</ul><br />
</div><br />
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<br />
<h2>Attributions</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Strains NEBTurbo, BL21 (DE3) KO20, BL21 AI were provided by INSERM U1001.</li><br />
<li>Plasmids pET Duet, pACYC Duet, pACYC zmSIR, pACYC soFD zmSIR, pCDF FNR were provided by INSERM U1001.</li><br />
</ul><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Genes msSirA, msFprA, msFdxA were synthesized by IDT.</li><br />
<li>Project was designed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell and Edwin Wintermute. All experiments and modelling were performed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell. </li><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/Project/TargetTeam:Paris Bettencourt/Project/Target2013-10-29T03:46:40Z<p>Yzegman: </p>
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<img src="https://static.igem.org/mediawiki/2013/3/3a/PB_logoParis.gif" width="122px" style="position:absolute;top:40px;right:30px;"/><br />
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<img src="https://static.igem.org/mediawiki/2013/c/c7/PB_targettitle.png" style="margin-bottom:15px"/><br />
<div class="overbox"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li><br />
<li>Identified a potential anti-TB activity of Pyridoxine at high doses.</li><br />
</ul><br />
<p></p><br />
</div><br />
<div class="biocriks"><br />
<h2>BioBricks</h2><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li> <br />
<li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li><br />
</ol><br />
</div><br />
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<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria</p><br />
</div><br />
<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
</div><br />
</a><br />
<a href="#Model"><br />
<div class="hlink"><br />
<h2>Skip to Modeling</h2><br />
</div><br />
</a><br />
<a href="#Design"><br />
<div class="hlink"><br />
<h2>Skip to Design</h2><br />
</div><br />
</a><br />
<a href="#Results"> <br />
<div class="hlink" style="margin-right:0px"><br />
<h2>Skip to Results</h2><br />
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</a><br />
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</div><br />
<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SirA is essential for <i>M. tuberculosis</i> persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced <a href="#Reference">(Pinto <i>et al</i> 2007)</a>. Currently, there are no drug candidates that are known to specifically inhibit SirA and conventional drug screens involve do not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. We designed a working drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA. To this end we cloned Ito <i>E. coli </i><span style="font-style: normal;">the sulfite reduction pathway</span> of <i>M. smegmatis</i>, a non-pathogenic mycobacterial relative of <i>M. Tuberculosis</i>. Our model overcomes the problem of long doubling time of <i>M. tuberculosis</i>. Specific inhibition of the sulfite reduction pathway is scored by comparing a drug screen of our <i>E. coli</i> construct <i>vs.</i> wild-type. Any drug candidates that have activity against both the wild-type <i>E. coli</i> and our construct are non-specific inhibitors of <i>E. coli</i> growth. However, any drug candidates that inhibit only the growth of our <i>E. coli </i>construct will be <span style="font-style: normal;">SirA</span><i> </i><span style="font-style: normal;">pathway specific.</span> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" width="535px"/></a><br />
<p><b>Figure 1: Overview of Targeted Drug Screen Design</b></p><br />
</div><br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We used an <i>E. coli</i> model (iJR904) obtained from the <a href="http://bigg.ucsd.edu/bigg/main.pl">BiGG database</a> as a starting model to obtain wild-type growth rate (f = 0.9129 divisions/hour). We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f= -8e-13=0 divisions/hour indicating that the sulphite reduction pathway is essential for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth with the mycobacteria pathway reverts the growth phenotype back to wild-type levels (f = 0.9105 divisions/hour). We then wanted to expand our model to find new pathways that we could utilize for a targeted drug screen approach. We wrote a matlab script that finds all the essential reactions in <i>M. tuberculosis</i> and all the essential reactions in <i>E. coli</i>, and then tries to complement the essential reactions in the <i>E. coli</i> model with the essential reactions from <i>M. tuberculosis</i>. The model identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 metabolic reactions</a> that we could target. Additionally, due to the modular nature of the model, it can be used to find target-able metabolic reactions in any SBML file. The Matlab scripts can be found <a href="https://2013.igem.org/File:TargetFBA.zip">here</a> and requires <a href="http://opencobra.sourceforge.net/openCOBRA/Welcome.html">Cobra Toolbox 2.0</a> to function. Please visit the FBA page for a detailed list of <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">results</a>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" width="267.5px"/></a></center><br />
<p><b>Figure 2: Biomass Flux through <i>E. coli</i> and mycoSIR E. coli</b><div style="font-size: 90%">Flux balance analysis was run using Cobra Toolbox 2.0 on <i>E. coli</i> sbml model iJR904 with and without SULR reaction. Additionally an <i>E. coli</i> sbml model was built with the SULR reaction replaced with a reaction representing the mycobacterial SirA reaction and FprA reaction, as well as ferredoxin FdxA as an additional species. The Biomass flux is restored to 99.75% of the wild-type level with the synthetic mycobacterial system.</div></p><br />
</div><br />
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<br />
<div id="Model"></div><br />
<h2>Structural Analysis of SirA</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Superimposing the structures of <i>M.tuberculosis</i> SirA and <i>E.coli </i> CysI reveals high homology, in particular of the active sites. Both proteins have the same symmetry (psuedo 2 fold) indicative of a common evolutionary origin. Our analysis highlighted important conserved residues, involved in substrate binding to be Arg97, Arg130, Arg166, Lys207. These positively charged residues are conserved in the sulphite/nitrite reductase family. In addition, 4 Cys residues are conserved for iron-sulphur binding. </p><br />
<p>The most profound structural differences between the two enzymes are found in the ferredoxin binding site and SirA's most C terminal residues and several surface loop regions due to deletions or insertions. A stark difference is a covalent bond formed between Cys161 (thiolate) and Tyr69 (C carbon atom) found adjacent to the redox center (Cu ions) in SirA. The covalently bound residues act as a secondary cofactor in tyrosyl radical stabilization. </p> <br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" width="267.5px"/></a></center><br />
<p><b>Figure 3: The superimposed 3D protein structures of SirA and CysI.</b><div style="font-size: 90%"> 303 amino acids are involved in superimposition with an rsmd of 1.41Å. All domains and loops of CysI are coloured purple, whilst SirA is coloured according to structural similarity with CysI: Red indicates poor alignment whilst blue indicates good alignment.</div></p><br />
</div><br />
<br />
<div id="Model"></div><br />
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<h2>Identification of potential drug target binding sites</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Our structural analysis provided the basis for our drug target prediction. Using Chembl and swiss pdb, we have shown a predicted drug target site. Our calculation gives strong favour for a drug to be effective at this site. The calculation reflects the suitability of small molecules to the binding site under the Lipinski's Rule of 5.</p><br />
<p>The drug target is located at the interface of the three domains. This binding pocket exhibits a dense hydrophobic region. Our analysis targets 48 amino acids of SirA within 6Å of a modelled small drug molecule. Of these residues, only 6 amino acids are charged: His409, Asp453, Asp474, His500, Asp504 and Arg541.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" width="267.5px"/></a></center><br />
<p><b>Figure 4 Drug target locations in SirA </b><div style="font-size: 90%">A domain located in SirA, identified as a drug target through Chembl analysis.</div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Structure based pharmacophore modelling of mycobacterial Fpra</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Using LigandScount 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Fpra. Our search revealed Riboflavin (Vitamin B2) and Pyridoxine to be drug targets for Fpra. We used NADP interacting with the active site as the model of the pharmacore. Results showed pyridoxin to be a competitive inhibitor to NADP. Pyridoxin is a synthetic compound currently available as a prescribed drug. </p><br />
<p>Chembl analysis of Pyridoxine (vitamin B6) show that it's properties fulfill Lipinski's criteria of being an orally active drug in humans. These properties state that any small drug molecule must have: no more than 5 H bond donors, no more 10 H bond acceptors (N or O atoms), mol mass of less than 500 dalts and octanol-water partition coefficient log P of no greater than 5).</p> <br />
<p>We have shown the proposed properties of Pyridoxine's interaction with Fpra as a competitive inhibitor to NADP at Fpra's active site. The key amino acids at the active site are Ala205, GLN204 and Thr208. GLN204 and Ala205 act as hydrogen bond acceptors whilst Thr208 interacts with a H via van der waals forces. Pyridoxin is a smaller, more lipid soluble molecule than NADP, thus more fitting to Lipinski's criteria. </p> <br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" width="100%"/></a></center><br />
<p><b>Figure 5: </b><div style="font-size: 90%">Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div></p><br />
<center><a href="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" width="100%"/></a></center><br />
<p><b>Figure 6:</b></b><div style="font-size: 90%"> Comparison of NADP interaction with Fpra's active site and Pyridoxine's interaction to it's active site.</div> </p><br />
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</div><br />
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<br />
<div id="Design"></div><br />
<h2>Synthetic Mycobacteria Pathway</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
We designed a synthetic <i>M.smegmatis-</i> derived sulfite reduction pathway containing sirA - the sulfite reductase, and two supporting genes that are required for its function in <i>E.coli</i>: fdxA and fprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The genes' sequences were taken from previous work describing their expression <a href="#Reference">(Pinto <i>et al</i> 2007)</a> in <i>E.coli</i> for purification and in vitro characterization; we removed restriction sites and codon optimized for expression in <i>E. coli</i>. The genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genesand were transformed into our knock-out mutant strains of <i>E. coli</i>. Data on Growth curves can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Notebook#target_Monday_30th_September.html">here</a>. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" width="535px"/></a><br />
<p><b>Figure 7: Growth curves of <i>E. coli</i> mycoSIR</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MycoSIR <i>E. coli</i> (red). No growth was detected for uninduced MycoSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Creation of Knock out Mutants</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We prepared two strains of <i>E. coli</i> which have the sulfite reduction pathway deleted: BL21 (DE3) <i>ΔCysI Δfpr ΔydbK</i> and BL21 (AI) <i>ΔCysI</i>. CysI is responsible for sulfite reduction in <i>E. coli</i>, while <i>fpr and ydbK</i> are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to<i> E. coli</i> in which it is NADPH dependant. These genes were also removed to ensure that they do not interfere with our system. <br />
</div><br />
<div class="rightparagraph"><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Synthetic Corn Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group <a href="#Reference">(2011 Barstow et al)</a>. In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" width="535px"/></a><br />
<p><b>Figure 8:</b> Growth curves of <i>E. coli</i> maizeSIR<div style="font-size: 90%"> BL21 (DE3) ΔcysI containing the MaizeSIR pathway (MaizeSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MaizeSIR <i>E. coli</i> (red). No growth was detected for uninduced MaizeSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Results"></div><br />
<h2>Results</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Upon successful cloning of the three genes into our <i>E. coli</i> deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in <i>M. tuberculosis</i> and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in <i>E. coli</i>.<br />
</p><br />
</br><br />
<p><b>Figure 9:</b> Growth of zmSIR <i>E. coli</i> on minimal media. <div style="font-size: 90%">BL21 (DE3) ΔcysI cells transformed with 1, 2 and 3 genes of the 3-gene zmSIR synthetic pathway were grown for 24 hours on minimal media supplemented with 25 uM IPTG (see methods), along with a WT BL21 (DE3) serving as a negative control, and an untransformed BL21 (DE3) ΔcysI, as negative control. Rescue of growth required all genes of the synthetic pathway (SIR, FNR and FD). </div></p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" width="535px"/></a><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-score</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
The Z-score is a statistical measurement aimed at assessing the "hit effect" in a drug screen high throughput screening. It is a commonly used measurement that shows how well did the drug effect the growth of the assay strain and how significant is the decrease in growth.</p><br />
<p><br />
To calculate the Z-score we used our experimental <i>E. coli</i> strain BL21 (AI) ΔcysI that carries all three genes of the synthetic pathway (sirA, fprA, fdxA). We grew it in the M9 minimal media supplemented with amino acid sulfur dropout powder, in a 96 well plate. Four of the wells were "spiked" with antibiotics (Amp, Gent, Kan, and Spect). </p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
This served as a simulation of the drug screen without the actual drug library. Only the drug screen controls are used: growth in M9 as a negative control (no drugs) and growth in M9 + antibiotics as a positive control (a sure hit). We then compared the distribution of the growth (OD) in the negative control with the distribution of growth (OD) in the positive control. The Z-score shows the distance of the negative control mean from the positive control mean in negative control standard deviation units.</p><br />
<p><b>Our Z-score is: -10.2.</b></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-factor</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is a measurement complementary to the Z-score. It measures the assay's quality based on the same data extracted from the same experiment made for the Z-score. This calculation gives an estimation of how far the negative controls are from the positive controls. It is a comparison of the two distributions which assumes that both distributions are normal and calculate how far 99% of the data points of each distribution are from each other.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is given on a scale from 0 to 1. Scores between 0.5 and 1 show that the assay is good and will enable testing in High throughput screens.</p><br />
<p><b>Our Z-factor score is 0.58.</b></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2> MycoSir growth assays reveal the potential anti TB activity of Pyridoxine </h2><br />
<br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;</p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;</p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div class="leftparagraph"> <br />
<center><a href="https://static.igem.org/mediawiki/2013/d/d5/PB_RiboflavinData.png"><img width="80%" src="https://static.igem.org/mediawiki/2013/d/d5/PB_RiboflavinData.png"/></a></center><br />
<p><b>Figure 10:</b>Riboflavin has no effect on the growth of WT or synthetic MycoSIR <i>E. coli.</i><div style="font-size: 90%"></div> The indicated quantities of riboflavin were dissolved in water and added to cultures of WT or MycoSIR E. coli in 4 biological replicates. No significant growth effects were observed. </p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/8/86/PyridoxineData.png"><img width="80%" src="https://static.igem.org/mediawiki/2013/8/86/PyridoxineData.png"></a></center><br />
<p><b>Figure 11:</b> MycoSIR E. coli growth assays reveal a potential anti-TB acitivity of pyridoxine at high doses. <div style="font-size: 90%"> The indicated quantities of pyridoxine were dissolved in water and added to cultures of WT or MycoSIR E. coli in 4 biological replicates. Both strains were grown in defined minimal media, where MycoSIR E. coli require our synthetic pathway for growth. Low pyridoxine doses had no detectable effects. However, a very high dose of pyridoxine (10 mg/mL) substantially inhibited the growth of MycoSIR E. coli yet showed no effect on WT growth. This suggests pyridoxine specifically inhibits the activity of the Mycobacterial SirA pathway. Derivativization or other methods could be used to further enhance the affinity and specificity of this compound.</div> </p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Reference"></div><br />
<h2>Literature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Global Alliance for TB Drug Development, Tuberculosis. Scientific blueprint for tuberculosis drug development, Tuberculosis (Edinb) 81 Suppl 1, 1–52 (2001).</li><br />
<br />
<li>World Health Organization, Global Tuberculosis Report 2012 (2012).</li><br />
<br />
<li>K. Raman, K. Yeturu, N. Chandra, targetTB: A target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis, BMC Syst Biol 2, 109 (2008).</li><br />
<br />
<li>R. Pinto, J. S. Harrison, T. Hsu, W. R. Jacobs, T. S. Leyh, Sulfite Reduction in Mycobacteria, Journal of Bacteriology 189, 6714–6722 (2007).</li><br />
<br />
<li>B. Barstow C. M. Agapakis, P. M. Boyle, G. Grandl, P. A. Silver, E. H. Wintermute, A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism, J Biol Eng 5, 7 (2011).</li><br />
</ul><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ. 2011 Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6:1290-1307.</li><br />
<br />
<li>Schellenberger, J., Park, J. O., Conrad, T. C., and Palsson, B. Ø., BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions, BMC Bioinformatics, 11:213, (2010).</li><br />
<br />
<li>S. G. Franzblau et al., Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis, Tuberculosis 92, 453–488 (2012).</li><br />
<br />
<li>D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Drugs for bad bugs: confronting the challenges of antibacterial discovery, Nat Rev Drug Discov 6, 29–40 (2006).</li><br />
<br />
<li>M. Nakayama, T. Akashi, T. Hase, Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin, J. Inorg. Biochem. 82, 27–32 (2000).</li><br />
</ul><br />
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<h2>Attributions</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Strains NEBTurbo, BL21 (DE3) KO20, BL21 AI were provided by INSERM U1001.</li><br />
<li>Plasmids pET Duet, pACYC Duet, pACYC zmSIR, pACYC soFD zmSIR, pCDF FNR were provided by INSERM U1001.</li><br />
</ul><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Genes msSirA, msFprA, msFdxA were synthesized by IDT.</li><br />
<li>Project was designed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell and Edwin Wintermute. All experiments and modelling were performed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell. </li><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-29T03:37:44Z<p>Yzegman: </p>
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<a href="#Detect"><br />
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<h2>Detect</h2><br />
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<a href="#Target"><br />
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<h2>Target</h2><br />
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<h2>Infiltrate</h2><br />
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<h2>Sabotage</h2><br />
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<a href="#Modelling"> <br />
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<h2>Modelling</h2><br />
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<a href="#Human_Practice"><br />
<div class="hlink" style="width:355px;margin-right:0"><br />
<h2>Human Practice</h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
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<div class="biocriks"><br />
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<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
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<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant <i>E. coli</i>. </b>We introduced our CRISPR-based DNA cleavage system to two strains of <i>E. coli</i> : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT <i>E. coli</i> could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR <i>E. coli</i> could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
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<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
<li>Identified a potential anti-TB activity of Pyridoxine at high doses.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
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<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
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<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR <i>E. coli</i> depend on our synthetic pathway for growth.</b> <i>E. coli</i> strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
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<div class="subbox2"><br />
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<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the listeriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
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</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed <i>E. coli</i> and WT <i>M.smegmatis</i> in LB media. Plating assays were used to count specifically <i>M.smegmatis</i> after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced <i>E. coli</i> (blue line) were stable.<br />
</p><br />
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<div id="Sabotage"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically burden of a device is critical for the maintenance of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of <i>E. coli</i> to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
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<center><br> <img width="70%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the propagation of horizontal gene-transfer via phagemid/helper system. We study the effect of the burden of a desired device on the maintenance of the system in time.</p><br />
</br><br />
<center><a href="https://static.igem.org/mediawiki/2013/3/38/PB_Model_diagram3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/3/38/PB_Model_diagram3.png" width="80%"/></a></center><br />
</br><br />
<p style="font-size:13px">Left: scheme representing the regular non-lytic M13 bacteriophage horizontal spread. Right: scheme representing the main processes of the phagemid/helper system.</p> <br />
</div><br />
<div class="projtile" style=""><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structure Based Modelling</a></h2><br />
<p><b>SirA: </b>Using Swiss pdb we demonstrated the superimposed 3D structures of <i>Mycobacterium tuberculosis</i> SirA and <i>Escherichia coli</i> CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
<br />
<p><b>FprA: </b>Using LigandScout 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Ferredoxin NADP reductase (FprA). We have modelled Pyridoxine's interaction to its active site. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an <i>E. coli</i> model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction which encodes for the sulphite reduction pathway and obtained a f value of -8.63e-13 indicating that the sulphite reduction pathway is essential. We wrote a program which finds all essential reactions in <i>M. tuberculosis</i> and <i>E. coli</i> SBML models and attempts to restore growth for each essential <i>E. coli</i> model with essential reactions from M. tuberculosis to identify other metabolic pathways we could apply a targeted drug screen to. We identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 other essential reactions</a> we can target.</p><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
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<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p >A comprehensive and quantitative study of gender (in)equality in iGEM and synthetic biology. A database was gathered and statistically analysed in order to depict sex ratio of iGEM teams' students and supervisors.</p> <br />
<br><br />
<center><img width="45%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br><br />
<p style="margin-top:-10px;font-size:13px"><b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.</p><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
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<div class="projtile" style="height:300px"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile" style="height:300px"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-29T03:36:26Z<p>Yzegman: </p>
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<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
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<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
<style><br />
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<div style="height:68px"><br />
<a href="#Detect"><br />
<div class="hlink" style="width:355px"><br />
<h2>Detect</h2><br />
</div><br />
</a><br />
<a href="#Target"><br />
<div class="hlink" style="width:355px"><br />
<h2>Target</h2><br />
</div><br />
</a><br />
<a href="#Infiltrate"><br />
<div class="hlink" style="width:355px;margin-right:0"><br />
<h2>Infiltrate</h2><br />
</div><br />
</a><br />
<div style="clear: both;"></div><br />
</div><br />
<div style="height:68px"><br />
<a href="#Sabotage"><br />
<div class="hlink" style="width:355px"><br />
<h2>Sabotage</h2><br />
</div><br />
</a><br />
<a href="#Modelling"> <br />
<div class="hlink" style="width:355px"><br />
<h2>Modelling</h2><br />
</div><br />
</a><br />
<a href="#Human_Practice"><br />
<div class="hlink" style="width:355px;margin-right:0"><br />
<h2>Human Practice</h2><br />
</div><br />
</a><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div id="page"><br />
<div id="Detect"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
<li>Identified a potential anti-TB activity of Pyridoxine at high doses.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant <i>E. coli</i>. </b>We introduced our CRISPR-based DNA cleavage system to two strains of <i>E. coli</i> : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT <i>E. coli</i> could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR <i>E. coli</i> could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR <i>E. coli</i> depend on our synthetic pathway for growth.</b> <i>E. coli</i> strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Infiltrate"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the listeriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed <i>E. coli</i> and WT <i>M.smegmatis</i> in LB media. Plating assays were used to count specifically <i>M.smegmatis</i> after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced <i>E. coli</i> (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
<div id="Sabotage"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically burden of a device is critical for the maintenance of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of <i>E. coli</i> to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="70%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
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<br />
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<br />
<div id="Modelling"></id><br />
<h2><a href="">Modelling</a></h2><br />
<div class="overbox" style=""><br />
<div class="projtile" style=""><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the propagation of horizontal gene-transfer via phagemid/helper system. We study the effect of the burden of a desired device on the maintenance of the system in time.</p><br />
</br><br />
<center><a href="https://static.igem.org/mediawiki/2013/3/38/PB_Model_diagram3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/3/38/PB_Model_diagram3.png" width="80%"/></a></center><br />
</br><br />
<p style="font-size:13px">Left: scheme representing the regular non-lytic M13 bacteriophage horizontal spread. Right: scheme representing the main processes of the phagemid/helper system.</p> <br />
</div><br />
<div class="projtile" style=""><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structure Based Modelling</a></h2><br />
<p><b>SirA: </b>Using Swiss pdb we demonstrated the superimposed 3D structures of <i>Mycobacterium tuberculosis</i> SirA and <i>Escherichia coli</i> CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
<br />
<p><b>FprA: </b>Using LigandScout 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Ferredoxin NADP reductase (FprA). We have modelled Pyridoxine's interaction to its active site. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an <i>E. coli</i> model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction which encodes for the sulphite reduction pathway and obtained a f value of -8.63e-13 indicating that the sulphite reduction pathway is essential. We wrote a program which finds all essential reactions in <i>M. tuberculosis</i> and <i>E. coli</i> SBML models and attempts to restore growth for each essential <i>E. coli</i> model with essential reactions from M. tuberculosis to identify other metabolic pathways we could apply a targeted drug screen to. We identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 other essential reactions</a> we can target.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Human_Practice"></id><br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
<style><br />
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<div class="overbox"><br />
<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p >A comprehensive and quantitative study of gender (in)equality in iGEM and synthetic biology. A database was gathered and statistically analysed in order to depict sex ratio of iGEM teams' students and supervisors.</p> <br />
<br><br />
<center><img width="45%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br><br />
<p style="margin-top:-10px;font-size:13px"><b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.</p><br />
<br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Collaboration"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox" style="height:300px"><br />
<div class="projtile" style="height:300px"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile" style="height:300px"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="height:300px;margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/Project/TargetTeam:Paris Bettencourt/Project/Target2013-10-29T03:28:54Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
<html><br />
<br />
<div id="page"><br />
<div style="width:1100px;margin:0 auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3a/PB_logoParis.gif" width="122px" style="position:absolute;top:40px;right:30px;"/><br />
</div><br />
<img src="https://static.igem.org/mediawiki/2013/c/c7/PB_targettitle.png" style="margin-bottom:15px"/><br />
<div class="overbox"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li><br />
</ul><br />
<p></p><br />
</div><br />
<div class="biocriks"><br />
<h2>BioBricks</h2><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li> <br />
<li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li><br />
</ol><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria</p><br />
</div><br />
<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
</div><br />
</a><br />
<a href="#Model"><br />
<div class="hlink"><br />
<h2>Skip to Modeling</h2><br />
</div><br />
</a><br />
<a href="#Design"><br />
<div class="hlink"><br />
<h2>Skip to Design</h2><br />
</div><br />
</a><br />
<a href="#Results"> <br />
<div class="hlink" style="margin-right:0px"><br />
<h2>Skip to Results</h2><br />
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</div><br />
<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SirA is essential for <i>M. tuberculosis</i> persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced <a href="#Reference">(Pinto <i>et al</i> 2007)</a>. Currently, there are no drug candidates that are known to specifically inhibit SirA and conventional drug screens involve do not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. We designed a working drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA. To this end we cloned Ito <i>E. coli </i><span style="font-style: normal;">the sulfite reduction pathway</span> of <i>M. smegmatis</i>, a non-pathogenic mycobacterial relative of <i>M. Tuberculosis</i>. Our model overcomes the problem of long doubling time of <i>M. tuberculosis</i>. Specific inhibition of the sulfite reduction pathway is scored by comparing a drug screen of our <i>E. coli</i> construct <i>vs.</i> wild-type. Any drug candidates that have activity against both the wild-type <i>E. coli</i> and our construct are non-specific inhibitors of <i>E. coli</i> growth. However, any drug candidates that inhibit only the growth of our <i>E. coli </i>construct will be <span style="font-style: normal;">SirA</span><i> </i><span style="font-style: normal;">pathway specific.</span> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" width="535px"/></a><br />
<p><b>Figure 1: Overview of Targeted Drug Screen Design</b></p><br />
</div><br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We used an <i>E. coli</i> model (iJR904) obtained from the <a href="http://bigg.ucsd.edu/bigg/main.pl">BiGG database</a> as a starting model to obtain wild-type growth rate (f = 0.9129 divisions/hour). We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f= -8e-13=0 divisions/hour indicating that the sulphite reduction pathway is essential for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth with the mycobacteria pathway reverts the growth phenotype back to wild-type levels (f = 0.9105 divisions/hour). We then wanted to expand our model to find new pathways that we could utilize for a targeted drug screen approach. We wrote a matlab script that finds all the essential reactions in <i>M. tuberculosis</i> and all the essential reactions in <i>E. coli</i>, and then tries to complement the essential reactions in the <i>E. coli</i> model with the essential reactions from <i>M. tuberculosis</i>. The model identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 metabolic reactions</a> that we could target. Additionally, due to the modular nature of the model, it can be used to find target-able metabolic reactions in any SBML file. The Matlab scripts can be found <a href="https://2013.igem.org/File:TargetFBA.zip">here</a> and requires <a href="http://opencobra.sourceforge.net/openCOBRA/Welcome.html">Cobra Toolbox 2.0</a> to function. Please visit the FBA page for a detailed list of <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">results</a>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" width="267.5px"/></a></center><br />
<p><b>Figure 2: Biomass Flux through <i>E. coli</i> and mycoSIR E. coli</b><div style="font-size: 90%">Flux balance analysis was run using Cobra Toolbox 2.0 on <i>E. coli</i> sbml model iJR904 with and without SULR reaction. Additionally an <i>E. coli</i> sbml model was built with the SULR reaction replaced with a reaction representing the mycobacterial SirA reaction and FprA reaction, as well as ferredoxin FdxA as an additional species. The Biomass flux is restored to 99.75% of the wild-type level with the synthetic mycobacterial system.</div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Model"></div><br />
<h2>Structural Analysis of SirA</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Superimposing the structures of <i>M.tuberculosis</i> SirA and <i>E.coli </i> CysI reveals high homology, in particular of the active sites. Both proteins have the same symmetry (psuedo 2 fold) indicative of a common evolutionary origin. Our analysis highlighted important conserved residues, involved in substrate binding to be Arg97, Arg130, Arg166, Lys207. These positively charged residues are conserved in the sulphite/nitrite reductase family. In addition, 4 Cys residues are conserved for iron-sulphur binding. </p><br />
<p>The most profound structural differences between the two enzymes are found in the ferredoxin binding site and SirA's most C terminal residues and several surface loop regions due to deletions or insertions. A stark difference is a covalent bond formed between Cys161 (thiolate) and Tyr69 (C carbon atom) found adjacent to the redox center (Cu ions) in SirA. The covalently bound residues act as a secondary cofactor in tyrosyl radical stabilization. </p> <br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" width="267.5px"/></a></center><br />
<p><b>Figure 3: The superimposed 3D protein structures of SirA and CysI.</b><div style="font-size: 90%"> 303 amino acids are involved in superimposition with an rsmd of 1.41Å. All domains and loops of CysI are coloured purple, whilst SirA is coloured according to structural similarity with CysI: Red indicates poor alignment whilst blue indicates good alignment.</div></p><br />
</div><br />
<br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<h2>Identification of potential drug target binding sites</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Our structural analysis provided the basis for our drug target prediction. Using Chembl and swiss pdb, we have shown a predicted drug target site. Our calculation gives strong favour for a drug to be effective at this site. The calculation reflects the suitability of small molecules to the binding site under the Lipinski's Rule of 5.</p><br />
<p>The drug target is located at the interface of the three domains. This binding pocket exhibits a dense hydrophobic region. Our analysis targets 48 amino acids of SirA within 6Å of a modelled small drug molecule. Of these residues, only 6 amino acids are charged: His409, Asp453, Asp474, His500, Asp504 and Arg541.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" width="267.5px"/></a></center><br />
<p><b>Figure 4 Drug target locations in SirA </b><div style="font-size: 90%">A domain located in SirA, identified as a drug target through Chembl analysis.</div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Structure based pharmacophore modelling of mycobacterial Fpra</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Using LigandScount 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Fpra. Our search revealed Riboflavin (Vitamin B2) and Pyridoxine to be drug targets for Fpra. We used NADP interacting with the active site as the model of the pharmacore. Results showed pyridoxin to be a competitive inhibitor to NADP. Pyridoxin is a synthetic compound currently available as a prescribed drug. </p><br />
<p>Chembl analysis of Pyridoxine (vitamin B6) show that it's properties fulfill Lipinski's criteria of being an orally active drug in humans. These properties state that any small drug molecule must have: no more than 5 H bond donors, no more 10 H bond acceptors (N or O atoms), mol mass of less than 500 dalts and octanol-water partition coefficient log P of no greater than 5).</p> <br />
<p>We have shown the proposed properties of Pyridoxine's interaction with Fpra as a competitive inhibitor to NADP at Fpra's active site. The key amino acids at the active site are Ala205, GLN204 and Thr208. GLN204 and Ala205 act as hydrogen bond acceptors whilst Thr208 interacts with a H via van der waals forces. Pyridoxin is a smaller, more lipid soluble molecule than NADP, thus more fitting to Lipinski's criteria. </p> <br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" width="100%"/></a></center><br />
<p><b>Figure 5: </b><div style="font-size: 90%">Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div></p><br />
<center><a href="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" width="100%"/></a></center><br />
<p><b>Figure 6:</b> Comparison of NADP interaction with Fpra's active site and Pyridoxine's interaction to it's active site.</b><div style="font-size: 90%"> Description Here</div> </p><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Design"></div><br />
<h2>Synthetic Mycobacteria Pathway</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
We designed a synthetic <i>M.smegmatis-</i> derived sulfite reduction pathway containing sirA - the sulfite reductase, and two supporting genes that are required for its function in <i>E.coli</i>: fdxA and fprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The genes' sequences were taken from previous work describing their expression <a href="#Reference">(Pinto <i>et al</i> 2007)</a> in <i>E.coli</i> for purification and in vitro characterization; we removed restriction sites and codon optimized for expression in <i>E. coli</i>. The genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genesand were transformed into our knock-out mutant strains of <i>E. coli</i>. Data on Growth curves can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Notebook#target_Monday_30th_September.html">here</a>. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" width="535px"/></a><br />
<p><b>Figure 5: Growth curves of <i>E. coli</i> mycoSIR</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MycoSIR <i>E. coli</i> (red). No growth was detected for uninduced MycoSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Creation of Knock out Mutants</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We prepared two strains of <i>E. coli</i> which have the sulfite reduction pathway deleted: BL21 (DE3) <i>ΔCysI Δfpr ΔydbK</i> and BL21 (AI) <i>ΔCysI</i>. CysI is responsible for sulfite reduction in <i>E. coli</i>, while <i>fpr and ydbK</i> are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to<i> E. coli</i> in which it is NADPH dependant. These genes were also removed to ensure that they do not interfere with our system. <br />
</div><br />
<div class="rightparagraph"><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Synthetic Corn Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group <a href="#Reference">(2011 Barstow et al)</a>. In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" width="535px"/></a><br />
<p><b>Figure 6: Growth curves of <i>E. coli</i> maizeSIR</b><div style="font-size: 90%"> BL21 (DE3) ΔcysI containing the MaizeSIR pathway (MaizeSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MaizeSIR <i>E. coli</i> (red). No growth was detected for uninduced MaizeSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
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<div id="Results"></div><br />
<h2>Results</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Upon successful cloning of the three genes into our <i>E. coli</i> deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in <i>M. tuberculosis</i> and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" width="535px"/></a><br />
<p><b>Figure 7: Growth of zmSIR <i>E. coli</i> on minimal media.</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI cells transformed with 1, 2 and 3 genes of the 3-gene zmSIR synthetic pathway were grown for 24 hours on minimal media supplemented with 25 uM IPTG (see methods), along with a WT BL21 (DE3) serving as a negative control, and an untransformed BL21 (DE3) ΔcysI, as negative control. Rescue of growth required all genes of the synthetic pathway (SIR, FNR and FD). </div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-score</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
The Z-score is a statistical measurement aimed at assessing the "hit effect" in a drug screen high throughput screening. It is a commonly used measurement that shows how well did the drug effect the growth of the assay strain and how significant is the decrease in growth.</p><br />
<p><br />
To calculate the Z-score we used our experimental <i>E. coli</i> strain BL21 (AI) ΔcysI that carries all three genes of the synthetic pathway (sirA, fprA, fdxA). We grew it in the M9 minimal media supplemented with amino acid sulfur dropout powder, in a 96 well plate. Four of the wells were "spiked" with antibiotics (Amp, Gent, Kan, and Spect). </p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
This served as a simulation of the drug screen without the actual drug library. Only the drug screen controls are used: growth in M9 as a negative control (no drugs) and growth in M9 + antibiotics as a positive control (a sure hit). We then compared the distribution of the growth (OD) in the negative control with the distribution of growth (OD) in the positive control. The Z-score shows the distance of the negative control mean from the positive control mean in negative control standard deviation units.</p><br />
<p><b>Our Z-score is: -10.2.</b></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-factor</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is a measurement complementary to the Z-score. It measures the assay's quality based on the same data extracted from the same experiment made for the Z-score. This calculation gives an estimation of how far the negative controls are from the positive controls. It is a comparison of the two distributions which assumes that both distributions are normal and calculate how far 99% of the data points of each distribution are from each other.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is given on a scale from 0 to 1. Scores between 0.5 and 1 show that the assay is good and will enable testing in High throughput screens.</p><br />
<p><b>Our Z-factor score is 0.58.</b></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div class="rightparagraph"><br />
<h2> MycoSir growth assays reveal the potential anti TB activity of Pyridoxine </h2><br />
<img width="70%" src="https://static.igem.org/mediawiki/2013/d/d5/PB_RiboflavinData.png"><br />
<br><p>Description</p><br><br />
<img width="70%" src="https://static.igem.org/mediawiki/2013/8/86/PyridoxineData.png"><br />
<br><p>Description</p><br />
<p>&nbsp;&nbsp;<br />
text.</p><br />
<p><b>Our Z-factor score is 0.58.</b></p><br />
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<br />
<br />
<br />
<div id="Reference"></div><br />
<h2>Literature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Global Alliance for TB Drug Development, Tuberculosis. Scientific blueprint for tuberculosis drug development, Tuberculosis (Edinb) 81 Suppl 1, 1–52 (2001).</li><br />
<br />
<li>World Health Organization, Global Tuberculosis Report 2012 (2012).</li><br />
<br />
<li>K. Raman, K. Yeturu, N. Chandra, targetTB: A target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis, BMC Syst Biol 2, 109 (2008).</li><br />
<br />
<li>R. Pinto, J. S. Harrison, T. Hsu, W. R. Jacobs, T. S. Leyh, Sulfite Reduction in Mycobacteria, Journal of Bacteriology 189, 6714–6722 (2007).</li><br />
<br />
<li>B. Barstow C. M. Agapakis, P. M. Boyle, G. Grandl, P. A. Silver, E. H. Wintermute, A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism, J Biol Eng 5, 7 (2011).</li><br />
</ul><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ. 2011 Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6:1290-1307.</li><br />
<br />
<li>Schellenberger, J., Park, J. O., Conrad, T. C., and Palsson, B. Ø., BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions, BMC Bioinformatics, 11:213, (2010).</li><br />
<br />
<li>S. G. Franzblau et al., Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis, Tuberculosis 92, 453–488 (2012).</li><br />
<br />
<li>D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Drugs for bad bugs: confronting the challenges of antibacterial discovery, Nat Rev Drug Discov 6, 29–40 (2006).</li><br />
<br />
<li>M. Nakayama, T. Akashi, T. Hase, Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin, J. Inorg. Biochem. 82, 27–32 (2000).</li><br />
</ul><br />
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<br />
<h2>Attributions</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Strains NEBTurbo, BL21 (DE3) KO20, BL21 AI were provided by INSERM U1001.</li><br />
<li>Plasmids pET Duet, pACYC Duet, pACYC zmSIR, pACYC soFD zmSIR, pCDF FNR were provided by INSERM U1001.</li><br />
</ul><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Genes msSirA, msFprA, msFdxA were synthesized by IDT.</li><br />
<li>Project was designed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell and Edwin Wintermute. All experiments and modelling were performed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell. </li><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/Project/TargetTeam:Paris Bettencourt/Project/Target2013-10-29T03:25:07Z<p>Yzegman: </p>
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<div style="width:1100px;margin:0 auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3a/PB_logoParis.gif" width="122px" style="position:absolute;top:40px;right:30px;"/><br />
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<img src="https://static.igem.org/mediawiki/2013/c/c7/PB_targettitle.png" style="margin-bottom:15px"/><br />
<div class="overbox"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li><br />
</ul><br />
<p></p><br />
</div><br />
<div class="biocriks"><br />
<h2>BioBricks</h2><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li> <br />
<li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li><br />
</ol><br />
</div><br />
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<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria</p><br />
</div><br />
<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
</div><br />
</a><br />
<a href="#Model"><br />
<div class="hlink"><br />
<h2>Skip to Modeling</h2><br />
</div><br />
</a><br />
<a href="#Design"><br />
<div class="hlink"><br />
<h2>Skip to Design</h2><br />
</div><br />
</a><br />
<a href="#Results"> <br />
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<h2>Skip to Results</h2><br />
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<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SirA is essential for <i>M. tuberculosis</i> persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced <a href="#Reference">(Pinto <i>et al</i> 2007)</a>. Currently, there are no drug candidates that are known to specifically inhibit SirA and conventional drug screens involve do not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. We designed a working drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA. To this end we cloned Ito <i>E. coli </i><span style="font-style: normal;">the sulfite reduction pathway</span> of <i>M. smegmatis</i>, a non-pathogenic mycobacterial relative of <i>M. Tuberculosis</i>. Our model overcomes the problem of long doubling time of <i>M. tuberculosis</i>. Specific inhibition of the sulfite reduction pathway is scored by comparing a drug screen of our <i>E. coli</i> construct <i>vs.</i> wild-type. Any drug candidates that have activity against both the wild-type <i>E. coli</i> and our construct are non-specific inhibitors of <i>E. coli</i> growth. However, any drug candidates that inhibit only the growth of our <i>E. coli </i>construct will be <span style="font-style: normal;">SirA</span><i> </i><span style="font-style: normal;">pathway specific.</span> <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" width="535px"/></a><br />
<p><b>Figure 1: Overview of Targeted Drug Screen Design</b></p><br />
</div><br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We used an <i>E. coli</i> model (iJR904) obtained from the <a href="http://bigg.ucsd.edu/bigg/main.pl">BiGG database</a> as a starting model to obtain wild-type growth rate (f = 0.9129 divisions/hour). We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f= -8e-13=0 divisions/hour indicating that the sulphite reduction pathway is essential for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth with the mycobacteria pathway reverts the growth phenotype back to wild-type levels (f = 0.9105 divisions/hour). We then wanted to expand our model to find new pathways that we could utilize for a targeted drug screen approach. We wrote a matlab script that finds all the essential reactions in <i>M. tuberculosis</i> and all the essential reactions in <i>E. coli</i>, and then tries to complement the essential reactions in the <i>E. coli</i> model with the essential reactions from <i>M. tuberculosis</i>. The model identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 metabolic reactions</a> that we could target. Additionally, due to the modular nature of the model, it can be used to find target-able metabolic reactions in any SBML file. The Matlab scripts can be found <a href="https://2013.igem.org/File:TargetFBA.zip">here</a> and requires <a href="http://opencobra.sourceforge.net/openCOBRA/Welcome.html">Cobra Toolbox 2.0</a> to function. Please visit the FBA page for a detailed list of <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">results</a>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" width="267.5px"/></a></center><br />
<p><b>Figure 2: Biomass Flux through <i>E. coli</i> and mycoSIR E. coli</b><div style="font-size: 90%">Flux balance analysis was run using Cobra Toolbox 2.0 on <i>E. coli</i> sbml model iJR904 with and without SULR reaction. Additionally an <i>E. coli</i> sbml model was built with the SULR reaction replaced with a reaction representing the mycobacterial SirA reaction and FprA reaction, as well as ferredoxin FdxA as an additional species. The Biomass flux is restored to 99.75% of the wild-type level with the synthetic mycobacterial system.</div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Model"></div><br />
<h2>Structural Analysis of SirA</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Superimposing the structures of <i>M.tuberculosis</i> SirA and <i>E.coli </i> CysI reveals high homology, in particular of the active sites. Both proteins have the same symmetry (psuedo 2 fold) indicative of a common evolutionary origin. Our analysis highlighted important conserved residues, involved in substrate binding to be Arg97, Arg130, Arg166, Lys207. These positively charged residues are conserved in the sulphite/nitrite reductase family. In addition, 4 Cys residues are conserved for iron-sulphur binding. </p><br />
<p>The most profound structural differences between the two enzymes are found in the ferredoxin binding site and SirA's most C terminal residues and several surface loop regions due to deletions or insertions. A stark difference is a covalent bond formed between Cys161 (thiolate) and Tyr69 (C carbon atom) found adjacent to the redox center (Cu ions) in SirA. The covalently bound residues act as a secondary cofactor in tyrosyl radical stabilization. </p> <br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" width="267.5px"/></a></center><br />
<p><b>Figure 3: The superimposed 3D protein structures of SirA and CysI.</b><div style="font-size: 90%"> 303 amino acids are involved in superimposition with an rsmd of 1.41Å. All domains and loops of CysI are coloured purple, whilst SirA is coloured according to structural similarity with CysI: Red indicates poor alignment whilst blue indicates good alignment.</div></p><br />
</div><br />
<br />
<div id="Model"></div><br />
<div style="clear: both;"></div><br />
<h2>Identification of potential drug target binding sites</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Our structural analysis provided the basis for our drug target prediction. Using Chembl and swiss pdb, we have shown a predicted drug target site. Our calculation gives strong favour for a drug to be effective at this site. The calculation reflects the suitability of small molecules to the binding site under the Lipinski's Rule of 5.</p><br />
<p>The drug target is located at the interface of the three domains. This binding pocket exhibits a dense hydrophobic region. Our analysis targets 48 amino acids of SirA within 6Å of a modelled small drug molecule. Of these residues, only 6 amino acids are charged: His409, Asp453, Asp474, His500, Asp504 and Arg541.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" width="267.5px"/></a></center><br />
<p><b>Figure 4 Drug target locations in SirA </b><div style="font-size: 90%">A domain located in SirA, identified as a drug target through Chembl analysis.</div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Structure based pharmacophore modelling of mycobacterial Fpra</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Using LigandScount 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Fpra. Our search revealed Riboflavin (Vitamin B2) and Pyridoxine to be drug targets for Fpra. We used NADP interacting with the active site as the model of the pharmacore. Results showed pyridoxin to be a competitive inhibitor to NADP. Pyridoxin is a synthetic compound currently available as a prescribed drug. </p><br />
<p>Chembl analysis of Pyridoxine (vitamin B6) show that it's properties fulfill Lipinski's criteria of being an orally active drug in humans. These properties state that any small drug molecule must have: no more than 5 H bond donors, no more 10 H bond acceptors (N or O atoms), mol mass of less than 500 dalts and octanol-water partition coefficient log P of no greater than 5).</p> <br />
<p>We have shown the proposed properties of Pyridoxine's interaction with Fpra as a competitive inhibitor to NADP at Fpra's active site. The key amino acids at the active site are Ala205, GLN204 and Thr208. GLN204 and Ala205 act as hydrogen bond acceptors whilst Thr208 interacts with a H via van der waals forces. Pyridoxin is a smaller, more lipid soluble molecule than NADP, thus more fitting to Lipinski's criteria. </p> <br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" width="100%"/></a></center><br />
<p><b>Figure 5: </b><div style="font-size: 90%">Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div></p><br />
<center><a href="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" width="100%"/></a></center><br />
<p><b>Figure 6:</b> Comparison of NADP interaction with Fpra's active site and Pyridoxine's interaction to it's active site.</b><div style="font-size: 90%"> Description Here</div> </p><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Design"></div><br />
<h2>Synthetic Mycobacteria Pathway</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
We designed a synthetic <i>M.smegmatis-</i> derived sulfite reduction pathway containing sirA - the sulfite reductase, and two supporting genes that are required for its function in <i>E.coli</i>: fdxA and fprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The genes' sequences were taken from previous work describing their expression <a href="#Reference">(Pinto <i>et al</i> 2007)</a> in <i>E.coli</i> for purification and in vitro characterization; we removed restriction sites and codon optimized for expression in <i>E. coli</i>. The genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genesand were transformed into our knock-out mutant strains of <i>E. coli</i>. Data on Growth curves can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Notebook#target_Monday_30th_September.html">here</a>. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" width="535px"/></a><br />
<p><b>Figure 5: Growth curves of <i>E. coli</i> mycoSIR</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MycoSIR <i>E. coli</i> (red). No growth was detected for uninduced MycoSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Creation of Knock out Mutants</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We prepared two strains of <i>E. coli</i> which have the sulfite reduction pathway deleted: BL21 (DE3) <i>ΔCysI Δfpr ΔydbK</i> and BL21 (AI) <i>ΔCysI</i>. CysI is responsible for sulfite reduction in <i>E. coli</i>, while <i>fpr and ydbK</i> are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to<i> E. coli</i> in which it is NADPH dependant. These genes were also removed to ensure that they do not interfere with our system. <br />
</div><br />
<div class="rightparagraph"><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Synthetic Corn Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group <a href="#Reference">(2011 Barstow et al)</a>. In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" width="535px"/></a><br />
<p><b>Figure 6: Growth curves of <i>E. coli</i> maizeSIR</b><div style="font-size: 90%"> BL21 (DE3) ΔcysI containing the MaizeSIR pathway (MaizeSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MaizeSIR <i>E. coli</i> (red). No growth was detected for uninduced MaizeSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Results"></div><br />
<h2>Results</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Upon successful cloning of the three genes into our <i>E. coli</i> deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in <i>M. tuberculosis</i> and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" width="535px"/></a><br />
<p><b>Figure 7: Growth of zmSIR <i>E. coli</i> on minimal media.</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI cells transformed with 1, 2 and 3 genes of the 3-gene zmSIR synthetic pathway were grown for 24 hours on minimal media supplemented with 25 uM IPTG (see methods), along with a WT BL21 (DE3) serving as a negative control, and an untransformed BL21 (DE3) ΔcysI, as negative control. Rescue of growth required all genes of the synthetic pathway (SIR, FNR and FD). </div></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-score</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
The Z-score is a statistical measurement aimed at assessing the "hit effect" in a drug screen high throughput screening. It is a commonly used measurement that shows how well did the drug effect the growth of the assay strain and how significant is the decrease in growth.</p><br />
<p><br />
To calculate the Z-score we used our experimental <i>E. coli</i> strain BL21 (AI) ΔcysI that carries all three genes of the synthetic pathway (sirA, fprA, fdxA). We grew it in the M9 minimal media supplemented with amino acid sulfur dropout powder, in a 96 well plate. Four of the wells were "spiked" with antibiotics (Amp, Gent, Kan, and Spect). </p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
This served as a simulation of the drug screen without the actual drug library. Only the drug screen controls are used: growth in M9 as a negative control (no drugs) and growth in M9 + antibiotics as a positive control (a sure hit). We then compared the distribution of the growth (OD) in the negative control with the distribution of growth (OD) in the positive control. The Z-score shows the distance of the negative control mean from the positive control mean in negative control standard deviation units.</p><br />
<p><b>Our Z-score is: -10.2.</b></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Z-factor</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is a measurement complementary to the Z-score. It measures the assay's quality based on the same data extracted from the same experiment made for the Z-score. This calculation gives an estimation of how far the negative controls are from the positive controls. It is a comparison of the two distributions which assumes that both distributions are normal and calculate how far 99% of the data points of each distribution are from each other.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is given on a scale from 0 to 1. Scores between 0.5 and 1 show that the assay is good and will enable testing in High throughput screens.</p><br />
<p><b>Our Z-factor score is 0.58.</b></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div class="rightparagraph"><br />
<img width="70%" src="https://static.igem.org/mediawiki/2013/d/d5/PB_RiboflavinData.png"><br />
<br><p>Description</p><br><br />
<img width="70%" src="https://static.igem.org/mediawiki/2013/8/86/PyridoxineData.png"><br />
<br><p>Description</p><br />
<p>&nbsp;&nbsp;<br />
text.</p><br />
<p><b>Our Z-factor score is 0.58.</b></p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
<br />
<div id="Reference"></div><br />
<h2>Literature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Global Alliance for TB Drug Development, Tuberculosis. Scientific blueprint for tuberculosis drug development, Tuberculosis (Edinb) 81 Suppl 1, 1–52 (2001).</li><br />
<br />
<li>World Health Organization, Global Tuberculosis Report 2012 (2012).</li><br />
<br />
<li>K. Raman, K. Yeturu, N. Chandra, targetTB: A target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis, BMC Syst Biol 2, 109 (2008).</li><br />
<br />
<li>R. Pinto, J. S. Harrison, T. Hsu, W. R. Jacobs, T. S. Leyh, Sulfite Reduction in Mycobacteria, Journal of Bacteriology 189, 6714–6722 (2007).</li><br />
<br />
<li>B. Barstow C. M. Agapakis, P. M. Boyle, G. Grandl, P. A. Silver, E. H. Wintermute, A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism, J Biol Eng 5, 7 (2011).</li><br />
</ul><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ. 2011 Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6:1290-1307.</li><br />
<br />
<li>Schellenberger, J., Park, J. O., Conrad, T. C., and Palsson, B. Ø., BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions, BMC Bioinformatics, 11:213, (2010).</li><br />
<br />
<li>S. G. Franzblau et al., Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis, Tuberculosis 92, 453–488 (2012).</li><br />
<br />
<li>D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Drugs for bad bugs: confronting the challenges of antibacterial discovery, Nat Rev Drug Discov 6, 29–40 (2006).</li><br />
<br />
<li>M. Nakayama, T. Akashi, T. Hase, Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin, J. Inorg. Biochem. 82, 27–32 (2000).</li><br />
</ul><br />
</div><br />
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<br />
<h2>Attributions</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Strains NEBTurbo, BL21 (DE3) KO20, BL21 AI were provided by INSERM U1001.</li><br />
<li>Plasmids pET Duet, pACYC Duet, pACYC zmSIR, pACYC soFD zmSIR, pCDF FNR were provided by INSERM U1001.</li><br />
</ul><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Genes msSirA, msFprA, msFdxA were synthesized by IDT.</li><br />
<li>Project was designed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell and Edwin Wintermute. All experiments and modelling were performed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell. </li><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/File:PyridoxineData.pngFile:PyridoxineData.png2013-10-29T03:18:22Z<p>Yzegman: </p>
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<div></div>Yzegmanhttp://2013.igem.org/File:PB_RiboflavinData.pngFile:PB RiboflavinData.png2013-10-29T03:17:37Z<p>Yzegman: uploaded a new version of &quot;File:PB RiboflavinData.png&quot;</p>
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<div></div>Yzegmanhttp://2013.igem.org/File:PB_HighSchool_Gender_Students.pngFile:PB HighSchool Gender Students.png2013-10-29T03:15:26Z<p>Yzegman: </p>
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<div></div>Yzegmanhttp://2013.igem.org/File:PB_RiboflavinData.pngFile:PB RiboflavinData.png2013-10-29T03:04:04Z<p>Yzegman: </p>
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<div></div>Yzegmanhttp://2013.igem.org/File:PB_PyridoxineData.pngFile:PB PyridoxineData.png2013-10-29T03:03:36Z<p>Yzegman: </p>
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<div></div>Yzegmanhttp://2013.igem.org/File:Zscore.pngFile:Zscore.png2013-10-29T03:00:40Z<p>Yzegman: </p>
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<div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/Project/TargetTeam:Paris Bettencourt/Project/Target2013-10-29T02:58:39Z<p>Yzegman: </p>
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<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
<html><br />
<br />
<div id="page"><br />
<div style="width:1100px;margin:0 auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3a/PB_logoParis.gif" width="122px" style="position:absolute;top:40px;right:30px;"/><br />
</div><br />
<img src="https://static.igem.org/mediawiki/2013/c/c7/PB_targettitle.png" style="margin-bottom:15px"/><br />
<div class="overbox"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an <i>E. coli</i> strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li><br />
<li>Demonstrated that <i>E. coli</i> can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li><br />
</ul><br />
<p></p><br />
</div><br />
<div class="biocriks"><br />
<h2>BioBricks</h2><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li> <br />
<li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li><br />
</ol><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria</p><br />
</div><br />
<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
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<div class="hlink"><br />
<h2>Skip to Modeling</h2><br />
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<a href="#Design"><br />
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<h2>Skip to Design</h2><br />
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<a href="#Results"> <br />
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<h2>Skip to Results</h2><br />
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<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SirA is essential for <i>M. tuberculosis</i> persistence phenotype as sulfur containing amino acids are particularly sensitive to oxidative stress within the macrophage and must regularly be replaced <a href="#Reference">(Pinto <i>et al</i> 2007)</a>. Currently, there are no drug candidates that are known to specifically inhibit SirA and conventional drug screens involve do not provide information regarding the mechanism of drug action nor do compounds that inhibit exponential growth necessarily have an effect on persistent TB. We designed a working drug screen assay to specifically target the mycobacterial sulfite reductase protein SirA. To this end we cloned Ito <i>E. coli </i><span style="font-style: normal;">the sulfite reduction pathway</span> of <i>M. smegmatis</i>, a non-pathogenic mycobacterial relative of <i>M. Tuberculosis</i>. Our model overcomes the problem of long doubling time of <i>M. tuberculosis</i>. Specific inhibition of the sulfite reduction pathway is scored by comparing a drug screen of our <i>E. coli</i> construct <i>vs.</i> wild-type. Any drug candidates that have activity against both the wild-type <i>E. coli</i> and our construct are non-specific inhibitors of <i>E. coli</i> growth. However, any drug candidates that inhibit only the growth of our <i>E. coli </i>construct will be <span style="font-style: normal;">SirA</span><i> </i><span style="font-style: normal;">pathway specific.</span> <br />
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<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/4f/PS_Drug_Scheme.png" width="535px"/></a><br />
<p><b>Figure 1: Overview of Targeted Drug Screen Design</b></p><br />
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<div id="Model"></div><br />
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<h2>Flux Balance Analysis of Sulfite Reduction Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We used an <i>E. coli</i> model (iJR904) obtained from the <a href="http://bigg.ucsd.edu/bigg/main.pl">BiGG database</a> as a starting model to obtain wild-type growth rate (f = 0.9129 divisions/hour). We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f= -8e-13=0 divisions/hour indicating that the sulphite reduction pathway is essential for growth. Finally we introduced two new reactions for sirA and fprA and a new species fdxA. We found that growth with the mycobacteria pathway reverts the growth phenotype back to wild-type levels (f = 0.9105 divisions/hour). We then wanted to expand our model to find new pathways that we could utilize for a targeted drug screen approach. We wrote a matlab script that finds all the essential reactions in <i>M. tuberculosis</i> and all the essential reactions in <i>E. coli</i>, and then tries to complement the essential reactions in the <i>E. coli</i> model with the essential reactions from <i>M. tuberculosis</i>. The model identified <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">100 metabolic reactions</a> that we could target. Additionally, due to the modular nature of the model, it can be used to find target-able metabolic reactions in any SBML file. The Matlab scripts can be found <a href="https://2013.igem.org/File:TargetFBA.zip">here</a> and requires <a href="http://opencobra.sourceforge.net/openCOBRA/Welcome.html">Cobra Toolbox 2.0</a> to function. Please visit the FBA page for a detailed list of <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target/FBA">results</a>.<br />
</p><br />
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<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/7/76/PS_FBA.png" width="267.5px"/></a></center><br />
<p><b>Figure 2: Biomass Flux through <i>E. coli</i> and mycoSIR E. coli</b><div style="font-size: 90%">Flux balance analysis was run using Cobra Toolbox 2.0 on <i>E. coli</i> sbml model iJR904 with and without SULR reaction. Additionally an <i>E. coli</i> sbml model was built with the SULR reaction replaced with a reaction representing the mycobacterial SirA reaction and FprA reaction, as well as ferredoxin FdxA as an additional species. The Biomass flux is restored to 99.75% of the wild-type level with the synthetic mycobacterial system.</div></p><br />
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<div id="Model"></div><br />
<h2>Structural Analysis of SirA</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Superimposing the structures of <i>M.tuberculosis</i> SirA and <i>E.coli </i> CysI reveals high homology, in particular of the active sites. Both proteins have the same symmetry (psuedo 2 fold) indicative of a common evolutionary origin. Our analysis highlighted important conserved residues, involved in substrate binding to be Arg97, Arg130, Arg166, Lys207. These positively charged residues are conserved in the sulphite/nitrite reductase family. In addition, 4 Cys residues are conserved for iron-sulphur binding. </p><br />
<p>The most profound structural differences between the two enzymes are found in the ferredoxin binding site and SirA's most C terminal residues and several surface loop regions due to deletions or insertions. A stark difference is a covalent bond formed between Cys161 (thiolate) and Tyr69 (C carbon atom) found adjacent to the redox center (Cu ions) in SirA. The covalently bound residues act as a secondary cofactor in tyrosyl radical stabilization. </p> <br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/52/Purplecys_SirA-1.png" width="267.5px"/></a></center><br />
<p><b>Figure 3: The superimposed 3D protein structures of SirA and CysI.</b><div style="font-size: 90%"> 303 amino acids are involved in superimposition with an rsmd of 1.41Å. All domains and loops of CysI are coloured purple, whilst SirA is coloured according to structural similarity with CysI: Red indicates poor alignment whilst blue indicates good alignment.</div></p><br />
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<div id="Model"></div><br />
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<h2>Identification of potential drug target binding sites</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Our structural analysis provided the basis for our drug target prediction. Using Chembl and swiss pdb, we have shown a predicted drug target site. Our calculation gives strong favour for a drug to be effective at this site. The calculation reflects the suitability of small molecules to the binding site under the Lipinski's Rule of 5.</p><br />
<p>The drug target is located at the interface of the three domains. This binding pocket exhibits a dense hydrophobic region. Our analysis targets 48 amino acids of SirA within 6Å of a modelled small drug molecule. Of these residues, only 6 amino acids are charged: His409, Asp453, Asp474, His500, Asp504 and Arg541.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/5/59/Drug_target_withoutAA.png" width="267.5px"/></a></center><br />
<p><b>Figure 4 Drug target locations in SirA </b><div style="font-size: 90%">A domain located in SirA, identified as a drug target through Chembl analysis.</div></p><br />
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<h2>Structure based pharmacophore modelling of mycobacterial Fpra</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Using LigandScount 3.1, we searched over 8100 drug compounds from the BindingDB and Chembl databases for drugs targetting mycobacterial Fpra. Our search revealed Riboflavin (Vitamin B2) and Pyridoxine to be drug targets for Fpra. We used NADP interacting with the active site as the model of the pharmacore. Results showed pyridoxin to be a competitive inhibitor to NADP. Pyridoxin is a synthetic compound currently available as a prescribed drug. </p><br />
<p>Chembl analysis of Pyridoxine (vitamin B6) show that it's properties fulfill Lipinski's criteria of being an orally active drug in humans. These properties state that any small drug molecule must have: no more than 5 H bond donors, no more 10 H bond acceptors (N or O atoms), mol mass of less than 500 dalts and octanol-water partition coefficient log P of no greater than 5).</p> <br />
<p>We have shown the proposed properties of Pyridoxine's interaction with Fpra as a competitive inhibitor to NADP at Fpra's active site. The key amino acids at the active site are Ala205, GLN204 and Thr208. GLN204 and Ala205 act as hydrogen bond acceptors whilst Thr208 interacts with a H via van der waals forces. Pyridoxin is a smaller, more lipid soluble molecule than NADP, thus more fitting to Lipinski's criteria. </p> <br />
</div><br />
<div class="rightparagraph"><br />
<center><a href="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/42/PB_Fnr_ribbons3.png" width="100%"/></a></center><br />
<p><b>Figure 5: </b><div style="font-size: 90%">Our 3D model shows the structure of FNR where negative residues are coloured in blue, positive residues in red and NAD in purple (ball and stick representation). The key amino acids at the active site are Glu211, Gly 366, Arg 110, Arg 199, Arg 200 and Asn155. Glu211 acts as a hydrogen acceptor whilst the latter four residues act as hydrogen donors.</div></p><br />
<center><a href="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/0/0a/PB_picture16.15.png" width="100%"/></a></center><br />
<p><b>Figure 6:</b> Comparison of NADP interaction with Fpra's active site and Pyridoxine's interaction to it's active site.</b><div style="font-size: 90%"> Description Here</div> </p><br />
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<div id="Design"></div><br />
<h2>Synthetic Mycobacteria Pathway</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
We designed a synthetic <i>M.smegmatis-</i> derived sulfite reduction pathway containing sirA - the sulfite reductase, and two supporting genes that are required for its function in <i>E.coli</i>: fdxA and fprA. FdxA is a mycobacterial Ferredoxin cofactor which is oxidised by SirA during the sulfite reduction reaction and FprA is a Ferredoxin-NADPH reductase use replenish the reduced Fdx pool. The genes' sequences were taken from previous work describing their expression <a href="#Reference">(Pinto <i>et al</i> 2007)</a> in <i>E.coli</i> for purification and in vitro characterization; we removed restriction sites and codon optimized for expression in <i>E. coli</i>. The genes were then cloned into two Duet expression vectors, one containing sirA and one containing the supporting genesand were transformed into our knock-out mutant strains of <i>E. coli</i>. Data on Growth curves can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Notebook#target_Monday_30th_September.html">here</a>. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/84/TB_drug_FinalSmeg.png" width="535px"/></a><br />
<p><b>Figure 5: Growth curves of <i>E. coli</i> mycoSIR</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MycoSIR <i>E. coli</i> (red). No growth was detected for uninduced MycoSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
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<h2>Creation of Knock out Mutants</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
We prepared two strains of <i>E. coli</i> which have the sulfite reduction pathway deleted: BL21 (DE3) <i>ΔCysI Δfpr ΔydbK</i> and BL21 (AI) <i>ΔCysI</i>. CysI is responsible for sulfite reduction in <i>E. coli</i>, while <i>fpr and ydbK</i> are two non-essential genes that consume ferredoxin. These two genes are deleted, as sulfite reduction in mycobacteria is ferredoxin dependent in comparison to<i> E. coli</i> in which it is NADPH dependant. These genes were also removed to ensure that they do not interfere with our system. <br />
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<div class="rightparagraph"><br />
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<h2>Synthetic Corn Pathway</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Additionally we prototyped the system with a reconstruction of a sulphite reduction pathway previously designed and published by the silver group <a href="#Reference">(2011 Barstow et al)</a>. In place of CysI, a corn (Zea mays) derived sulfite reductase (zmSIR) was used. Two additional genes were included: Spinach ferredoxin (soFD), and corn derived ferredoxin NADP+ reductase (zmFNR). These genes, respectively, are required for production of the ferredoxin cofactor and the NADP+ ferredoxin reductase and are required for sulfite reductase (zmSIR) to function within <i>E. coli</i>.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a2/PB_final_Corn.png" width="535px"/></a><br />
<p><b>Figure 6: Growth curves of <i>E. coli</i> maizeSIR</b><div style="font-size: 90%"> BL21 (DE3) ΔcysI containing the MaizeSIR pathway (MaizeSIR <i>E. coli</i>) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 <i>E. coli</i>, (blue), and the MaizeSIR <i>E. coli</i> (red). No growth was detected for uninduced MaizeSIR <i>E. coli</i> (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR <i>E. coli</i> and WT BL21 but not in uninduced zmSIR strain.</div></p> <br />
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<div id="Results"></div><br />
<h2>Results</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Upon successful cloning of the three genes into our <i>E. coli</i> deletion strain, we continued to confirm that all three genes are required for growth on minimal media. Our two synthetic pathways were found to rescue growth on a sulfurless amino acid supplemented minimal media. We hope that this technique of using synthetic biology to overcome problems faced in naturally occurring systems will be both a large boon to the pursuit of finding novel drug candidates in <i>M. tuberculosis</i> and more broadly as this technique can be used for high-throughput screening of any pathway that can be constructed to be essential for growth in <i>E. coli</i>.<br />
</p><br />
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<div class="rightparagraph"><br />
<a href="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/8/85/PS_D_Figure1_plate.png" width="535px"/></a><br />
<p><b>Figure 7: Growth of zmSIR <i>E. coli</i> on minimal media.</b> <div style="font-size: 90%">BL21 (DE3) ΔcysI cells transformed with 1, 2 and 3 genes of the 3-gene zmSIR synthetic pathway were grown for 24 hours on minimal media supplemented with 25 uM IPTG (see methods), along with a WT BL21 (DE3) serving as a negative control, and an untransformed BL21 (DE3) ΔcysI, as negative control. Rescue of growth required all genes of the synthetic pathway (SIR, FNR and FD). </div></p><br />
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<h2>Z-score</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
The Z-score is a statistical measurement aimed at assessing the "hit effect" in a drug screen high throughput screening. It is a commonly used measurement that shows how well did the drug effect the growth of the assay strain and how significant is the decrease in growth.</p><br />
<p><br />
To calculate the Z-score we used our experimental <i>E. coli</i> strain BL21 (AI) ΔcysI that carries all three genes of the synthetic pathway (sirA, fprA, fdxA). We grew it in the M9 minimal media supplemented with amino acid sulfur dropout powder, in a 96 well plate. Four of the wells were "spiked" with antibiotics (Amp, Gent, Kan, and Spect). </p><br />
</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
This served as a simulation of the drug screen without the actual drug library. Only the drug screen controls are used: growth in M9 as a negative control (no drugs) and growth in M9 + antibiotics as a positive control (a sure hit). We then compared the distribution of the growth (OD) in the negative control with the distribution of growth (OD) in the positive control. The Z-score shows the distance of the negative control mean from the positive control mean in negative control standard deviation units.</p><br />
<p><b>Our Z-score is: -10.2.</b></p><br />
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<br />
<h2>Z-factor</h2><br />
<div class="leftparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is a measurement complementary to the Z-score. It measures the assay's quality based on the same data extracted from the same experiment made for the Z-score. This calculation gives an estimation of how far the negative controls are from the positive controls. It is a comparison of the two distributions which assumes that both distributions are normal and calculate how far 99% of the data points of each distribution are from each other.</p><br />
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</div><br />
<div class="rightparagraph"><br />
<p>&nbsp;&nbsp;<br />
Z-factor is given on a scale from 0 to 1. Scores between 0.5 and 1 show that the assay is good and will enable testing in High throughput screens.</p><br />
<p><b>Our Z-factor score is 0.58.</b></p><br />
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<div id="Reference"></div><br />
<h2>Literature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Global Alliance for TB Drug Development, Tuberculosis. Scientific blueprint for tuberculosis drug development, Tuberculosis (Edinb) 81 Suppl 1, 1–52 (2001).</li><br />
<br />
<li>World Health Organization, Global Tuberculosis Report 2012 (2012).</li><br />
<br />
<li>K. Raman, K. Yeturu, N. Chandra, targetTB: A target identification pipeline for Mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis, BMC Syst Biol 2, 109 (2008).</li><br />
<br />
<li>R. Pinto, J. S. Harrison, T. Hsu, W. R. Jacobs, T. S. Leyh, Sulfite Reduction in Mycobacteria, Journal of Bacteriology 189, 6714–6722 (2007).</li><br />
<br />
<li>B. Barstow C. M. Agapakis, P. M. Boyle, G. Grandl, P. A. Silver, E. H. Wintermute, A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism, J Biol Eng 5, 7 (2011).</li><br />
</ul><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ. 2011 Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nature Protocols 6:1290-1307.</li><br />
<br />
<li>Schellenberger, J., Park, J. O., Conrad, T. C., and Palsson, B. Ø., BiGG: a Biochemical Genetic and Genomic knowledgebase of large scale metabolic reconstructions, BMC Bioinformatics, 11:213, (2010).</li><br />
<br />
<li>S. G. Franzblau et al., Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis, Tuberculosis 92, 453–488 (2012).</li><br />
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<li>D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Drugs for bad bugs: confronting the challenges of antibacterial discovery, Nat Rev Drug Discov 6, 29–40 (2006).</li><br />
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<li>M. Nakayama, T. Akashi, T. Hase, Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin, J. Inorg. Biochem. 82, 27–32 (2000).</li><br />
</ul><br />
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<h2>Attributions</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>Strains NEBTurbo, BL21 (DE3) KO20, BL21 AI were provided by INSERM U1001.</li><br />
<li>Plasmids pET Duet, pACYC Duet, pACYC zmSIR, pACYC soFD zmSIR, pCDF FNR were provided by INSERM U1001.</li><br />
</ul><br />
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<div class="rightparagraph"><br />
<ul><br />
<li>Genes msSirA, msFprA, msFdxA were synthesized by IDT.</li><br />
<li>Project was designed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell and Edwin Wintermute. All experiments and modelling were performed by Idonnya Aghoghogbe, Yonatan Zegman, Matthew Deyell. </li><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-29T02:31:54Z<p>Yzegman: </p>
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<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
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<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
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<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Infiltrate"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
<div id="Sabotage"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically burden of a device is critical for the maintenance of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="70%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
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<br />
<div id="Modelling"></id><br />
<h2><a href="">Modelling</a></h2><br />
<div class="overbox" style=""><br />
<div class="projtile" style=""><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the propagation of horizontal gene-transfer via phagemid/helper system. We study the effect of the burden of a desired device on the maintenance of the system in time.</p><br />
</br><br />
<center><a href="https://static.igem.org/mediawiki/2013/3/38/PB_Model_diagram3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/3/38/PB_Model_diagram3.png" width="80%"/></a></center><br />
</br><br />
<p style="font-size:13px">Left: scheme representing the regular non-lytic M13 bacteriophage horizontal spread. Right: scheme representing the main processes of the phagemid/helper system.</p> <br />
</div><br />
<div class="projtile" style=""><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of <i>Mycobacterium tuberculosis</i> SirA and <i>Escherichia coli</i> CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Human_Practice"></id><br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<div class="overbox"><br />
<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p >A comprehensive and quantitative study of gender (in)equality in iGEM and synthetic biology. A database was gathered and statistically analysed in order to depict sex ratio of iGEM teams' students and supervisors.</p> <br />
<br><br />
<center><img width="45%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br><br />
<p style="margin-top:-10px;font-size:13px"><b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.</p><br />
<br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div id="Collaboration"></id><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox" style="height:300px"><br />
<div class="projtile" style="height:300px"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile" style="height:300px"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="height:300px;margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/File:Newfiguresabotagepresentation.pngFile:Newfiguresabotagepresentation.png2013-10-29T02:30:52Z<p>Yzegman: uploaded a new version of &quot;File:Newfiguresabotagepresentation.png&quot;</p>
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<div></div>Yzegmanhttp://2013.igem.org/File:Bb_target_fdxA.pngFile:Bb target fdxA.png2013-10-29T01:54:47Z<p>Yzegman: uploaded a new version of &quot;File:Bb target fdxA.png&quot;</p>
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<div></div>Yzegmanhttp://2013.igem.org/File:Bb_target_fdxA.pngFile:Bb target fdxA.png2013-10-29T01:53:19Z<p>Yzegman: uploaded a new version of &quot;File:Bb target fdxA.png&quot;</p>
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<div></div>Yzegmanhttp://2013.igem.org/File:Newfiguresabotagepresentation.pngFile:Newfiguresabotagepresentation.png2013-10-29T01:03:44Z<p>Yzegman: uploaded a new version of &quot;File:Newfiguresabotagepresentation.png&quot;</p>
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<div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:37:54Z<p>Yzegman: </p>
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<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
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<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
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<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
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<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
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<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender (in)equality in iGEM and synthetic biology. A database was gathered and statistically analysed in order to depict sex ratio of iGEM teams' students and supervisors. <br />
<br><center><img width="50%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
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<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
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<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_StudyTeam:Paris Bettencourt/Human Practice/Gender Study2013-10-28T22:36:48Z<p>Yzegman: </p>
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<h2>Background</h2><br />
<p>Science suffers from gender bias</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Revealed gender bias in synthetic biology by studying sex ratios in SB conferences and labs</li><br />
<li>Built a database of all iGEM teams reporting all available online information and sex ratios of teams and advisors</li><br />
<li>Conducted a statistical analysis of this data-set and showed among other results that success in iGEM is correlated to gender mix</li><br />
<li>Made recommendations to implement an active gender policy in iGEM</li><br />
</ul><br />
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<div class="aims"><br />
<h2>Aims</h2><br />
<p>To investigate gender dynamics in iGEM and in synthetic biology research community at large in a quantitative manner</p><br />
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<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
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<a href="#Recommendations"><br />
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<h2>Skip to Recommendations</h2><br />
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<h2>Skip to Database</h2><br />
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<a href="#Findings"> <br />
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<h2>Skip to Main Findings</h2><br />
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<h2>Skip to Gender Bias in SynBio</h2><br />
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<a href="#Success"><br />
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<h2>Skip to iGEM Diversity and Success</h2><br />
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<h2>Skip to Clues to Improve Balance</h2><br />
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<a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Facts"><br />
<div class="hlink" style="width:100%;margin-right:0"><br />
<h2 style="font-size:22px;">Infographics on gender and Synthetic Biology</h2><br />
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<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
For every woman killed by TB, there are two men. Our review of the literature on gender bias and tuberculosis can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study/Gender_Bias">here</a>. If a disease can be biased, what about ourselves? iGEM? Synthetic biology? Gender bias in science may appear in different forms. Gender balance varies by discipline, by job title, by age or by region. Only 30% of researchers in Europe are women, while 92% of French university deans are men. </p> <p>Hisorically, gender bias has affected the lives of scientists and the practice of science. However, assessing gender bias today in a living community is very difficult. History, stereotypes, limits of the disciplines, and the simple lack of data can prevent us, the synthetic biologists, from thinking about our own relationship to gender.</p><br />
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</div><br />
<div class="rightparagraph"><br />
<p><br />
Most of those issues should not apply in synthetic biology. Synthetic biology is a new field. The argument of the heritage of some habits cannot be made. It is a mix of previously existing disciplines and therefore very open and should not reflect preexisting stereotypes. To study gender bias in iGEM and in synthetic biology we decided to follow a data driven approach. Studying in a quantitative manner this subjects had two main benefits. First it prevented us to apply our own biases and stereotypes on this subject. Secondly, it lead us to construct data base that we make freely available and let anyone test his own hypothesis on this controversial subject and form his own conclusions.</p><br />
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<div id="SynBio"></div><br />
<h2> Synthetic biology field : general overview of gender equality in synthetic biology </h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Gender repartition in synthetic biology can be looked at from different perspectives. For this study, two main ways were chosen: composition of labs and conferences. The main reasons for those choices were the accessibility of online data </p><br />
</div><br />
<div class="rightparagraph"><br />
<p><br />
as well as the necessity to get information not only about the general gender balance but also the sex ratio inside a defined category: PhD students, post docs, head of labs... </p><br />
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<br />
<h2> Synthetic biology labs, a good representation of gender (in)equality in science </h2> <br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Teams of 50 synthetic biology labs were studied. The labs were chosen by their presence on the webpage http://syntheticbiology.org/Labs.html . For each lab, several numbers were reported in a table : total number of people in the team, number of women in the team, number of PhD students, post docs, head of labs, number of women PhD students, post docs, head of labs. From this, the sex ratios (number of women / total number of people) were then calculated for each of those categories. </p><br />
<p><center><a href="https://2013.igem.org/File:ParisB2013_Synthetic_Biology_Research_Groups.xls">Download the database here</a></center></p> <br />
<p><br />
The first conclusion that can be made is that women are generally under-represented in synthetic biology labs. 33% correspond to the average presence of women in research in Europe. Indeed according to the European Commission, 32% of researchers in Europe are women <i>(She Figures, 2012)</i>. <br><br>The second finding also reflects well an already known reality in science : the glass ceiling. In 1995, the glass ceiling was defined by the U.S. Department of Labor, as a <i>"political term used to describe "the unseen, yet unbreakable barrier that keeps minorities and women from rising to the upper rungs of the corporate ladder, regardless of their qualifications or achievements" </i>.<br />
With only 17,85\% of heads of labs being women, synthetic biology is still doing slightly better than the average. According to a European study done in 2008 called <i>Mapping the maze,getting women to the top in research</i>., only 15% of women occupy top research position in Europe. However, the number of SB P.I. should be analyzed through the filter of history. In a new field, it would be expected in a world where bias would not be present anymore to have way more women at those positions.<br />
</p><br />
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</div><br />
<div class="rightparagraph"><br />
</br></br></br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/4/46/PB_GS_LabsBis.png" width="353px"/><br><br />
<b>Figure 1. </b> : Sex ratio in synthetic biology labs<br />
</center><br />
<br />
</br></br></br><br />
<center><br />
<TABLE BORDER="1"> <br />
<CAPTION> </CAPTION> <br />
<TR> <br />
<TH>Labs </TH> <br />
<TH> Phd Students </TH> <br />
<TH> Post Docs</TH> <br />
<TH> Head of Labs </TH> <br />
</TR> <br />
<TR> <br />
<TH> 33,10 % </TH> <br />
<TD> 35,39 % </TD> <br />
<TD> 31,31 % </TD> <br />
<TD> 17,85 % </TD> <br />
</TR> <br />
</TABLE><br />
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<br />
<h2> Speakers at SB Conferences : effects of an active gender policy</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SBX.0 conferences accompanied the development of synthetic biology. They provide a great way to investigate the evolution of gender ratio since the birth of synthetic biology. Moreover, the presence/absence of women as speakers is a known indicator of gender bias and specially of active gender policy. Indeed, several social mechanisms are in place lead to fewer female speakers that could be expected: self censorship, unconscious stereotypes, unconscious choice of only male speakers... However, having female speakers at conference is a key point. It allows women, to gain confidence but also to act as role model for women attending the conference. </p><br />
<p><br />
To study SB conferences, available programs online were downloaded. Data referring to the number of speakers but also to posters were recorded. The data-set could not be completed for certain years due to the impossibility of finding the data online.<br />
</p><br />
<p><center><a href="https://2013.igem.org/File:ParisB2013Resultats_SB.xls">Download the database here</a></center></p><br />
<p><br />
The sex ratio of the speakers have followed a very interesting evolution. It has been multiplied by 3 from SB1 to SB5. This could indicate a change of policy considering speakers. Most likely, the first conferences invited speakers without taking into consideration the gender dimension. Might it be due to some complaints or the raise in awareness of the conferences organizers, the numbers went up. This example is interesting because it clearly show an interest in the subject by the involved community.<br />
</p><br />
<p><br />
Two main conclusions can be drawn on posters. First, the sex ratio of authors in posters has changed throughout the years. Secondly, this number is not as high as the sex ratio in labs. The question is why? The points described above could be underlying reasons, however it is very difficult to truly go beyond this with only those numbers. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<br><br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/6/62/PB_GS_Sex_ratio_in_SB_Conf.png" width="400px"/><br><br />
<b> Figure 2. </b> Sex ratio in SB conferences<br />
</center><br />
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<br />
<h2> Under represented and badly represented</h2> <br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
In order to try to better understand the dynamics of gender behind the posters numbers, the rank of authors were reported for each poster. Sex ratio were calculated for each rank, keeping in mind that in biology, the first author is often a Phd student or a post doc and the last author, the P.I.<br />
</p><br />
<p><br />
As explained above, women are generally under-represented in synthetic biology labs, even less represented at conferences. When looking at the rank of author in posters, another bias appears. Indeed, women are more likely to be present as middle authors than first or last. This bias can be found in papers of different disciplines as shown on the graph realized on the eigenfactor.</p><br />
<p> <br />
The main finding considering gender in synthetic biology is that even though synthetic biology is new and interdisciplinary, it remains quite representative of existing gender bias in science. Therefore it can be concluded, that the issues that have kept women out of science and especially out of top research position are still present and will not be resolved with time. A strong and active policy appears necessary to bring more mixity and therefore diversity in this field.<br />
<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/PB_GS_Author_place_.png" width="435px"/> <br><br />
<b>Figure 3. </b> Sex ratio according to rank of authors in SB posters.<br />
</center><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/PB_GS_Eigenfactor.png" width="535px"/><br />
<b>Figure 4. </b> : Analysis of rank of authors according to gender in scientific publications<br />
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<div id="Database"></div><br />
<h2> iGEM as a model : a fantastic database </h2><br />
<div class="leftparagraph"><br />
<h3> Online Data</h3> <br />
<p> &nbsp;&nbsp;<br />
All the data concerning iGEM were retrieved from the website : <a href="https://igem.org">https://igem.org</a> <br />
List of teams were retrieved from the webpages <a href="https://igem.org/Team_List.cgi?year=2012">https://igem.org/Team_List.cgi?year=2012</a>.<br />
List of project themes were retrieved from <a href="https://igem.org/Team_Tracks?year=2012">https://igem.org/Team_Tracks?year=2012</a>.<br />
List of prices were retrieved <a href="https://igem.org/Results">https://igem.org/Results</a>.<br />
List of judges were retrieved from: <a href="https://igem.org/Judge_List">https://igem.org/Judge_List</a><br />
</p><br />
<h3> Sex ratio determination :</h3> <br />
<p> &nbsp;&nbsp; For each team, the official team profile was checked to count the number of student members, advisors and instructors.<br />
Then to determine the sex of particpants, wiki were used when names were not obvious, using pictures when they existed. When no pictures were available and names were not obviously referring to one sex, a google image search was done on the name (first and last name) and the sex was chosen as the most represented sex in the pictures (if 10 images of men come up and 30 of women, the participant was considered as a woman).</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<h3> Database : </h3> <br />
<p> &nbsp;&nbsp; Information for the first year of iGEM were difficult to find because of the non existence of available wiki pages and it was therefore decided not to take into account this year.<br />
Teams who withdrew during the competition were not taken into account since it was most of the time impossible to know the number of participants due to the absence of wiki.<br />
In the end our data set is composed of 662 teams over 5 years. For each team were reported : <br />
Year ; region ; name of the team ; number of student members ; number of women student members ; number of advisors ; number of women advisors ; number of instructors ; number of women instructors ; participation to MIT championship ; medal ; regional prices ; championship prices ;tracks. </p><br />
<p><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2013/3/35/PB_GS_IGEMdatabase.xls"> Download the database here</a><br />
</center><br />
</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Findings"></div><br />
<h2> iGEM : a mirror of main gender problems </h2><br />
<div class="leftparagraph"><br />
<h3> Teams sex ratio, a very robust value </h3> <br />
<p> &nbsp;&nbsp;<br />
<br><br><br />
The first thing that was examined was the evolution of sex ratio of teams in iGEM across continents and throughout the years.<br />
</p><br />
<br><br />
<p><br />
The striking conclusion of this comparison is that the sex ratio is iGEM teams remains constant through the years and across continents (ANOVA's p-value for the different conditions > 0,5). This shows that women are underrepresented in iGEM teams. </p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/5/5a/GS_Year.png" width="250px"/><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/GS_Region.png" width="250px"/><br />
<b>Figure 5. </b> Sex ratio of iGEM teams through the years and across continents<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="leftparagraph"><br />
<h3> Women do not supervise as much as men </h3> <br />
<p> &nbsp;&nbsp;<br />
The second question investigated was the sex ratios for the different categories of people participating in iGEM. Indeed, iGEM is not only undergrad students. Advisors, instructors, judges also participate representing the complete professional ladder of synthetic biology. A category called Supervisors was created corresponding to instructors and advisers. Indeed, those terms are not understood and used in the same way in different continents. In some countries "advisers" means people who directly teach the teams (mostly grad students and post docs) whereas it means general mentors for others and vice versa.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/GS_Role.png" width="300px" height="250px"/> <br><br />
<b>Figure 6. </b> Sex ratios in iGEM according to categories of people participating<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
When executing comparisons tests , team members' sex ratio is found to be different from judges' and instructors' ones (p value < 0,01). However judges and advisers are not significantly different ( p value > 0,5). This result reveals a tendency of women to supervise less than men. Indeed, from team members to instructors, the sex ratio is divided by two. What is even more interesting is to compare those numbers to sex ratios of PhD students and post docs in labs. The sex ratio of instructors is 10 points lower. </p><br />
<p> Women constitute a pool of talent that is not mobilized. They participate but do not supervise teams. They are "lost" along the way. Indeed, in a study published last year in PNAS, researchers showed that P.I. were less prone to have a woman mentoring students than man. This unconscious bias can be translated by a lack of encouragement from P.I.s but also by a self censorship which is not taken into account by other supervisors as explained in an recently published article by Eileen Pollack (E. Pollack Why Are There Still So Few Women in Science? NY TImes OCtober 2013).<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<h3> Tracks and sex ratio in iGEM </h3> <br />
<p> &nbsp;&nbsp;<br />
The third finding goes against an often-heard stereotype "women are more interested by applied research". In order to investigate this subject, tracks were reported for each project. In iGEM tracks correspond to general theme of the project : medicine, fundationnal research…Tracks were then looked at in terms of sex ratios. There is no significant difference between tracks. (ANOVA > 0,1).</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/PB_GS_Sexratiotracksbis.png" width="535px"/><br />
<b>Figure 7. </b> Sex ratios and tracks in iGEM<br />
</center><br />
<p><br />
To conclude, studying the iGEM competition gives a unique quantitative insight on existing questions in the field of gender studies. It also constitutes an amazing argument to convince scientists of the existence of a gender issue in science. As explained by Rascun et al in a recent paper published in PNAS, scientists believe that those type of bias only exist in some labs, not their own, therefore very objective studies need to be conducted to clearly show the reality of the numbers. More over , Jo Handelsman a microbiologist involved in that paper underlined in a recent interview, that people often think that there is still an issue in physics or maths but that there are no more women issues in biology, which is not true. This study supports strongly the view that this general thinking is untrue.<br />
</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Success"></div><br />
<h3> In iGEM, is diversity a factor of success ? </h3><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Several studies led by consulting groups (McKinsey and Company Women Matter, 2007) have shown that mixity in a team increases performance. The big question of what leads to success in iGEM was therefore investigated using the database with a special focus on gender. In order to be able to get a general idea about iGEM team success, a point system was put in place.<br />
</p><br />
<p>Points were attributed the following way.<br><br />
For the medal: 1 point for bronze medal, 2 points for silver medal, 3 points for gold medal. For the world jamboree qualified teams: 2 points for every team taking part in 2010 and before (before regional jamborees existed) , 6 points for team qualified for world final (after 2010). For special prices (Best ...): 6 points were attributed for each regional price earned (only after 2010), 13 points for each price earned in the world final (all price worth 13 points before regional jamborees existed). For the final place in world final: 15 points for the sixth team, 20 points for the fifth team, 25 points for the fourth team, 30 points for the third team, 35 points for the second team, 40 points for the firth team.</p><br />
<p><br />
The aim was to give each team a score that is proportional to the rewards it earned, taking in account that all teams were in world jamboree prior to 2011, without having to be qualified in regional jamborees.<br />
</p><br />
<p>Best score is for the Imperial College London team in 2011 (81 points).<br />
All teams (all years) average is 7.41 points, considering teams with no points (due to withdrew).</p><br />
<p><br />
Correlations studies between this number of points and other variables show that that for all teams, the main variables explaining success in iGEM is the number of years of existence and the size of the team. It would therefore seem that mixity would not be a factor. However, when looking at correlations between variables of teams who truly succeeded (points > 20) , the variables that have a significant correlation with the number of points become the sex ratio and the number of supervisors. Therefore it could be hypothesized that beginning iGEM teams have to face major challenges but when the team existed for a few years and general organization or funding problems have been dealt with , diversity could be a factor for success. </p><br />
<p><br />
In order to check if this could be seen in the best iGEM teams that existed, the sex ratio of of prize winner teams was compared to the one of participating teams with boostrap resampling giving a p-value of 0.035 This means that the sex ratio of winning teams (45%) is significantly different from the one of participating teams (37%)<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png" width="400px" ><br><br />
<b>Figure 8. </b> Gender balance and succes in iGEM<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Clues"></div><br />
<h2> Clues to improve mixity </h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
<br />
Women are not as represented as men in iGEM. Why should this be a problem ? Indeed, even if it might lead to success as explained above, the need to have gender equality could be questioned. However iGEM is an international competition. One of its main goals is to attract and educate young people as well as trying to have them solve real issues. Synthetic biology might be a key technology to solve the main challenges of the 21st century.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p> The world will need science and if iGEM only succeeds in motivating half of the population that could be interested, this would be a major failure to achieve its mission. Therefore, the last part of the study was aimed at understanding how could iGEM improve mixity within its own ranks.<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div class="leftparagraph"><br />
<h3> From the data </h3><br />
<p> &nbsp;&nbsp;<br />
By looking at correlation between sex ratios and other variables, the most striking result is the link between team size and sex ratio. Teams of 2 or 3 people are almost only male teams. Even when taking out those very small teams, out of the data set the correlation holds up. This is a first lead. <br><br />
The second analysis that was made regarding the data was to compare the detailed statistics of the 100 most female teams and 100 male teams. Again, it is found that the total team member is lower for male team (9,7 vs 7,8 (p-value 0,0019) we can hypothesize that having women instructors does matter to attract girls in teams. They serve as role models. Having a woman capable of studying and realizing a synthetic biology project is a direct signal to female students that it is also possible for them to do it. Having a woman adviser might also help girls better adapt in a group and reduce their fears about having to endure constant teasing or "male " ambiance.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<h3> From a survey</h3><br />
<p> &nbsp;&nbsp;<br />
Finally, a survey was conducted among iGEMers and former iGEMers to understand their motivations and activities in iGEM. The study was designed to be unbiased and to avoid stereotype threat (for example by putting the question about gender in the end among many other pieces of information). It is still available <a href="http://bit.ly/14WykuZ"> here</a>. Participants in the survey had to rank from 1 to 5 (1 being not important, and 5 very important) answers to questions regarding personnal and professional motivations for participating in iGEM as well values and on what did they spend their time. 63 people answered among whom 32% were women.</p><br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/c/c5/PB_GS_Survey1.png" width="500px" /><br />
<b>Figure 9. </b> Results of survey : what did you hope to learn in iGEM ?<br />
</center><br />
<br><br><br />
<p><br />
It is interesting to notice that men and women answered almost exactly the same way regarding most of the questions. Women gave a little more importance for the value of fundamental research in iGEM while men graded a bit better "Changing the world". Motivations were approximately the same as well as time spent on each activity. Just a little fact was that men considered human practices a bit more important than women did but spent a little less time on it. <br />
There is only one main difference (more than one point out of five which is represented below) : the will to lead a project and lead a team. It is striking to see how much men are more motivated to lead teams than women. This is definitely to put in relation with the number of women advisers found and the impact it can then have on teams mixity. This could reflect women lack of self esteem in some parts of their work.</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Recommendations"></div><br />
<h2> Recommendations</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Considering all the results that were presented above, here is a list of recommendations for the iGEM foundation to pursue an active policy to improve mixity in iGEM.<br />
<ul><br />
<li> Raise the number of women judges </li> <br />
<li> Promote large teams </li> <br />
<li> Write up a small paragraph to team heads to insist on the importance of motivating young women to be advisers.</li><br />
<li> Giving Bonus point when the team have women advisers </li><br />
</ul><br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<p>And finally, add in iGEM requirements a Gender reflection. By having teams filling out the database that was built and answering the survey and write a small paragraph about how they see mixity in their team and what it could bring, it would drastically raise the awareness of young men and women about the gender problem in science. Having an up-to-date database is also a great way to see improvements in a quantitative manner. It would allow a direct assessment of the effects of an active gender policy which would be a unique example in science. iGEM could become a leader in that fight and prepare the new generation of scientists to finally get rid of the gender inequality in science<br />
</p><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Litterature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>P. Allotey, M. Gyapong Gender in tuberculosis research INT J TUBERC LUNG DIS 2008 </li><br />
<li>M. Calid, S. Rasul, S Ullah Khan, M; Saeed Gender differences in delay to s to tuberculosis diagnosis and treatment outcome<br><br />
European Commission She figures 2012<br><br />
European Commission, Mapping the gaze : getting more women to the top in Research 2008.<br><br />
</li><br />
<li>C.B. Holmes, H. Hausler, P. Hunn : A review of sex differences in the epidemiology of tuberculosis</li><br />
<li>A. N. Martinez J. T. Rhee, P. M. Small,‡M. A. Behr Sex differences in the epidemiology of tuberculosis in San Francisco INT J TUBERC LUNG DIS 4(1):26–31 2000</li><br />
<li>Moss-Racusin et al, (2012) Science faculty’s subtle gender biases favor male students PNAS </li><br />
<br />
</ul><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>McKinsey and Company Women Matter, 2007 </li><br />
<li>Olivier Neyrolles, Lluis Quintana-Murci Sexual Inequality in Tuberculosis, Plos Medicine 2009</li><br />
<li>Nosek et al. (2009) National differences in gender–science stereotypes predict national sex differences in science and math achievement. PNAS June 30, 2009 vol. 106 no. 26 10593–10597</li><br />
<br />
<li>E. Pollack Why Are There Still So Few Women in Science? NY TImes OCtober 2013</li><br />
<li>Al S. Rhines The role of sex differences in the prevalence and transmission of tuberculosis : Tuberculosis 2013</li><br />
<li>M. W. Uplekar, S. Rangan, M. G. Weiss, J. Ogden, M. W. Borgdorff, P. Hudelson Attention to gender issues in tuberculosis control INT J TUBERC LUNG DIS 4(1):26–31 2001</li><br />
</ul><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Attributions</h2><br />
<p>We would like to thank Flora Vincent, President of <a href="http://wax-science.fr/")>WAX Science</a> association for her precious help in analyzing the results, and Kim de Mora and Kitwa from the iGEM foundation, for helping spreading the survey. <p><br />
<p>This project was designed and accomplished by Aude Bernheim, Clovis Basier, Matt Deyell, Marguerite Benony and Sebastian Jaramillo in consultation with Edwin Wintermute and Ariel Lindner.</p><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_StudyTeam:Paris Bettencourt/Human Practice/Gender Study2013-10-28T22:35:02Z<p>Yzegman: </p>
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<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Science suffers from gender bias</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Revealed gender bias in synthetic biology by studying sex ratios in SB conferences and labs</li><br />
<li>Built a database of all iGEM teams reporting all available online information and sex ratios of teams and advisors</li><br />
<li>Conducted a statistical analysis of this data-set and showed among other results that success in iGEM is correlated to gender mix</li><br />
<li>Made recommendations to implement an active gender policy in iGEM</li><br />
</ul><br />
<p></p><br />
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<div class="aims"><br />
<h2>Aims</h2><br />
<p>To investigate gender dynamics in iGEM and in synthetic biology research community at large in a quantitative manner</p><br />
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<div style="clear: both;"></div> <br />
<div style="height:68px"> <br />
<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
</div><br />
</a><br />
<a href="#Recommendations"><br />
<div class="hlink"><br />
<h2>Skip to Recommendations</h2><br />
</div><br />
</a><br />
<a href="#Database"><br />
<div class="hlink"><br />
<h2>Skip to Database</h2><br />
</div><br />
</a><br />
<a href="#Findings"> <br />
<div class="hlink" style="margin-right:0px"><br />
<h2>Skip to Main Findings</h2><br />
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<a href="#SynBio"><br />
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<h2>Skip to Gender Bias in SynBio</h2><br />
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</a><br />
<a href="#Success"><br />
<div class="hlink" style="width:355px"><br />
<h2>Skip to iGEM Diversity and Success</h2><br />
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</a><br />
<a href="#Clues"><br />
<div class="hlink" style="width:355px;margin-right:0"><br />
<h2>Skip to Clues to Improve Balance</h2><br />
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<a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Facts"><br />
<div class="hlink" style="width:100%;margin-right:0"><br />
<h2 style="font-size:22px;">Infographics on gender and Synthetic Biology</h2><br />
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<br />
<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
For every woman killed by TB, there are two men. Our review of the literature on gender bias and tuberculosis can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study/Gender_Bias">here</a>. If a disease can be biased, what about ourselves? iGEM? Synthetic biology? Gender bias in science may appear in different forms. Gender balance varies by discipline, by job title, by age or by region. Only 30% of researchers in Europe are women, while 92% of French university deans are men. </p> <p>Hisorically, gender bias has affected the lives of scientists and the practice of science. However, assessing gender bias today in a living community is very difficult. History, stereotypes, limits of the disciplines, and the simple lack of data can prevent us, the synthetic biologists, from thinking about our own relationship to gender.</p><br />
<br><br />
</div><br />
<div class="rightparagraph"><br />
<p><br />
Most of those issues should not apply in synthetic biology. Synthetic biology is a new field. The argument of the heritage of some habits cannot be made. It is a mix of previously existing disciplines and therefore very open and should not reflect preexisting stereotypes. To study gender bias in iGEM and in synthetic biology we decided to follow a data driven approach. Studying in a quantitative manner this subjects had two main benefits. First it prevented us to apply our own biases and stereotypes on this subject. Secondly, it lead us to construct data base that we make freely available and let anyone test his own hypothesis on this controversial subject and form his own conclusions.</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="SynBio"></div><br />
<h2> Synthetic biology field : general overview of gender equality in synthetic biology </h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Gender repartition in synthetic biology can be looked at from different perspectives. For this study, two main ways were chosen: composition of labs and conferences. The main reasons for those choices were the accessibility of online data </p><br />
</div><br />
<div class="rightparagraph"><br />
<p><br />
as well as the necessity to get information not only about the general gender balance but also the sex ratio inside a defined category: PhD students, post docs, head of labs... </p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2> Synthetic biology labs, a good representation of gender (in)equality in science </h2> <br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Teams of 50 synthetic biology labs were studied. The labs were chosen by their presence on the webpage http://syntheticbiology.org/Labs.html . For each lab, several numbers were reported in a table : total number of people in the team, number of women in the team, number of PhD students, post docs, head of labs, number of women PhD students, post docs, head of labs. From this, the sex ratios (number of women / total number of people) were then calculated for each of those categories. </p><br />
<p><center><a href="https://2013.igem.org/File:ParisB2013_Synthetic_Biology_Research_Groups.xls">Download the database here</a></center></p> <br />
<p><br />
The first conclusion that can be made is that women are generally under-represented in synthetic biology labs. 33% correspond to the average presence of women in research in Europe. Indeed according to the European Commission, 32% of researchers in Europe are women <i>(She Figures, 2012)</i>. <br><br>The second finding also reflects well an already known reality in science : the glass ceiling. In 1995, the glass ceiling was defined by the U.S. Department of Labor, as a <i>"political term used to describe "the unseen, yet unbreakable barrier that keeps minorities and women from rising to the upper rungs of the corporate ladder, regardless of their qualifications or achievements" </i>.<br />
With only 17,85\% of heads of labs being women, synthetic biology is still doing slightly better than the average. According to a European study done in 2008 called <i>Mapping the maze,getting women to the top in research</i>., only 15% of women occupy top research position in Europe. However, the number of SB P.I. should be analyzed through the filter of history. In a new field, it would be expected in a world where bias would not be present anymore to have way more women at those positions.<br />
</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
</br></br></br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/4/46/PB_GS_LabsBis.png" width="353px"/><br><br />
<b>Figure 1. </b> : Sex ratio in synthetic biology labs<br />
</center><br />
<br />
</br></br></br><br />
<center><br />
<TABLE BORDER="1"> <br />
<CAPTION> </CAPTION> <br />
<TR> <br />
<TH>Labs </TH> <br />
<TH> Phd Students </TH> <br />
<TH> Post Docs</TH> <br />
<TH> Head of Labs </TH> <br />
</TR> <br />
<TR> <br />
<TH> 33,10 % </TH> <br />
<TD> 35,39 % </TD> <br />
<TD> 31,31 % </TD> <br />
<TD> 17,85 % </TD> <br />
</TR> <br />
</TABLE><br />
</center><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2> Speakers at SB Conferences : effects of an active gender policy</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SBX.0 conferences accompanied the development of synthetic biology. They provide a great way to investigate the evolution of gender ratio since the birth of synthetic biology. Moreover, the presence/absence of women as speakers is a known indicator of gender bias and specially of active gender policy. Indeed, several social mechanisms are in place lead to fewer female speakers that could be expected: self censorship, unconscious stereotypes, unconscious choice of only male speakers... However, having female speakers at conference is a key point. It allows women, to gain confidence but also to act as role model for women attending the conference. </p><br />
<p><br />
To study SB conferences, available programs online were downloaded. Data referring to the number of speakers but also to posters were recorded. The data-set could not be completed for certain years due to the impossibility of finding the data online.<br />
</p><br />
<p><center><a href="https://2013.igem.org/File:ParisB2013Resultats_SB.xls">Download the database here</a></center></p><br />
<p><br />
The sex ratio of the speakers have followed a very interesting evolution. It has been multiplied by 3 from SB1 to SB5. This could indicate a change of policy considering speakers. Most likely, the first conferences invited speakers without taking into consideration the gender dimension. Might it be due to some complaints or the raise in awareness of the conferences organizers, the numbers went up. This example is interesting because it clearly show an interest in the subject by the involved community.<br />
</p><br />
<p><br />
Two main conclusions can be drawn on posters. First, the sex ratio of authors in posters has changed throughout the years. Secondly, this number is not as high as the sex ratio in labs. The question is why? The points described above could be underlying reasons, however it is very difficult to truly go beyond this with only those numbers. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<br><br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/6/62/PB_GS_Sex_ratio_in_SB_Conf.png" width="400px"/><br><br />
<b> Figure 2. </b> Sex ratio in SB conferences<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2> Under represented and badly represented</h2> <br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
In order to try to better understand the dynamics of gender behind the posters numbers, the rank of authors were reported for each poster. Sex ratio were calculated for each rank, keeping in mind that in biology, the first author is often a Phd student or a post doc and the last author, the P.I.<br />
</p><br />
<p><br />
As explained above, women are generally under-represented in synthetic biology labs, even less represented at conferences. When looking at the rank of author in posters, another bias appears. Indeed, women are more likely to be present as middle authors than first or last. This bias can be found in papers of different disciplines as shown on the graph realized on the eigenfactor.</p><br />
<p> <br />
The main finding considering gender in synthetic biology is that even though synthetic biology is new and interdisciplinary, it remains quite representative of existing gender bias in science. Therefore it can be concluded, that the issues that have kept women out of science and especially out of top research position are still present and will not be resolved with time. A strong and active policy appears necessary to bring more mixity and therefore diversity in this field.<br />
<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/PB_GS_Author_place_.png" width="435px"/> <br><br />
<b>Figure 3. </b> Sex ratio according to rank of authors in SB posters.<br />
</center><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/PB_GS_Eigenfactor.png" width="535px"/><br />
<b>Figure 4. </b> : Analysis of rank of authors according to gender in scientific publications<br />
</center> <br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Database"></div><br />
<h2> iGEM as a model : a fantastic database </h2><br />
<div class="leftparagraph"><br />
<h3> Online Data</h3> <br />
<p> &nbsp;&nbsp;<br />
All the data concerning iGEM were retrieved from the website : <a href="https://igem.org">https://igem.org</a> <br />
List of teams were retrieved from the webpages <a href="https://igem.org/Team_List.cgi?year=2012">https://igem.org/Team_List.cgi?year=2012</a>.<br />
List of project themes were retrieved from <a href="https://igem.org/Team_Tracks?year=2012">https://igem.org/Team_Tracks?year=2012</a>.<br />
List of prices were retrieved <a href="https://igem.org/Results">https://igem.org/Results</a>.<br />
List of judges were retrieved from: <a href="https://igem.org/Judge_List">https://igem.org/Judge_List</a><br />
</p><br />
<h3> Sex ratio determination :</h3> <br />
<p> &nbsp;&nbsp; For each team, the official team profile was checked to count the number of student members, advisors and instructors.<br />
Then to determine the sex of particpants, wiki were used when names were not obvious, using pictures when they existed. When no pictures were available and names were not obviously referring to one sex, a google image search was done on the name (first and last name) and the sex was chosen as the most represented sex in the pictures (if 10 images of men come up and 30 of women, the participant was considered as a woman).</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<h3> Database : </h3> <br />
<p> &nbsp;&nbsp; Information for the first year of iGEM were difficult to find because of the non existence of available wiki pages and it was therefore decided not to take into account this year.<br />
Teams who withdrew during the competition were not taken into account since it was most of the time impossible to know the number of participants due to the absence of wiki.<br />
In the end our data set is composed of 662 teams over 5 years. For each team were reported : <br />
Year ; region ; name of the team ; number of student members ; number of women student members ; number of advisors ; number of women advisors ; number of instructors ; number of women instructors ; participation to MIT championship ; medal ; regional prices ; championship prices ;tracks. </p><br />
<p><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2013/3/35/PB_GS_IGEMdatabase.xls"> Download the database here</a><br />
</center><br />
</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Findings"></div><br />
<h2> iGEM : a mirror of main gender problems </h2><br />
<div class="leftparagraph"><br />
<h3> Teams sex ratio, a very robust value </h3> <br />
<p> &nbsp;&nbsp;<br />
<br><br><br />
The first thing that was examined was the evolution of sex ratio of teams in iGEM across continents and throughout the years.<br />
</p><br />
<br><br />
<p><br />
The striking conclusion of this comparison is that the sex ratio is iGEM teams remains constant through the years and across continents (ANOVA's p-value for the different conditions > 0,5). This shows that women are underrepresented in iGEM teams. </p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/5/5a/GS_Year.png" width="250px"/><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/GS_Region.png" width="250px"/><br />
<b>Figure 5. </b> Sex ratio of iGEM teams through the years and across continents<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="leftparagraph"><br />
<h3> Women do not supervise as much as men </h3> <br />
<p> &nbsp;&nbsp;<br />
The second question investigated was the sex ratios for the different categories of people participating in iGEM. Indeed, iGEM is not only undergrad students. Advisors, instructors, judges also participate representing the complete professional ladder of synthetic biology. A category called Supervisors was created corresponding to instructors and advisers. Indeed, those terms are not understood and used in the same way in different continents. In some countries "advisers" means people who directly teach the teams (mostly grad students and post docs) whereas it means general mentors for others and vice versa.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/GS_Role.png" width="300px" height="250px"/> <br><br />
<b>Figure 6. </b> Sex ratios in iGEM according to categories of people participating<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
When executing comparisons tests , team members' sex ratio is found to be different from judges' and instructors' ones (p value < 0,01). However judges and advisers are not significantly different ( p value > 0,5). This result reveals a tendency of women to supervise less than men. Indeed, from team members to instructors, the sex ratio is divided by two. What is even more interesting is to compare those numbers to sex ratios of PhD students and post docs in labs. The sex ratio of instructors is 10 points lower. </p><br />
<p> Women constitute a pool of talent that is not mobilized. They participate but do not supervise teams. They are "lost" along the way. Indeed, in a study published last year in PNAS, researchers showed that P.I. were less prone to have a woman mentoring students than man. This unconscious bias can be translated by a lack of encouragement from P.I.s but also by a self censorship which is not taken into account by other supervisors as explained in an recently published article by Eileen Pollack (E. Pollack Why Are There Still So Few Women in Science? NY TImes OCtober 2013).<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<h3> Tracks and sex ratio in iGEM </h3> <br />
<p> &nbsp;&nbsp;<br />
The third finding goes against an often-heard stereotype "women are more interested by applied research". In order to investigate this subject, tracks were reported for each project. In iGEM tracks correspond to general theme of the project : medicine, fundationnal research…Tracks were then looked at in terms of sex ratios. There is no significant difference between tracks. (ANOVA > 0,1).</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/PB_GS_Sexratiotracksbis.png" width="535px"/><br />
<b>Figure 7. </b> Sex ratios and tracks in iGEM<br />
</center><br />
<p><br />
To conclude, studying the iGEM competition gives a unique quantitative insight on existing questions in the field of gender studies. It also constitutes an amazing argument to convince scientists of the existence of a gender issue in science. As explained by Rascun et al in a recent paper published in PNAS, scientists believe that those type of bias only exist in some labs, not their own, therefore very objective studies need to be conducted to clearly show the reality of the numbers. More over , Jo Handelsman a microbiologist involved in that paper underlined in a recent interview, that people often think that there is still an issue in physics or maths but that there are no more women issues in biology, which is not true. This study supports strongly the view that this general thinking is untrue.<br />
</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Success"></div><br />
<h3> In iGEM, is diversity a factor of success ? </h3><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Several studies led by consulting groups (McKinsey and Company Women Matter, 2007) have shown that mixity in a team increases performance. The big question of what leads to success in iGEM was therefore investigated using the database with a special focus on gender. In order to be able to get a general idea about iGEM team success, a point system was put in place.<br />
</p><br />
<p>Points were attributed the following way.<br><br />
For the medal: 1 point for bronze medal, 2 points for silver medal, 3 points for gold medal. For the world jamboree qualified teams: 2 points for every team taking part in 2010 and before (before regional jamborees existed) , 6 points for team qualified for world final (after 2010). For special prices (Best ...): 6 points were attributed for each regional price earned (only after 2010), 13 points for each price earned in the world final (all price worth 13 points before regional jamborees existed). For the final place in world final: 15 points for the sixth team, 20 points for the fifth team, 25 points for the fourth team, 30 points for the third team, 35 points for the second team, 40 points for the firth team.</p><br />
<p><br />
The aim was to give each team a score that is proportional to the rewards it earned, taking in account that all teams were in world jamboree prior to 2011, without having to be qualified in regional jamborees.<br />
</p><br />
<p>Best score is for the Imperial College London team in 2011 (81 points).<br />
All teams (all years) average is 7.41 points, considering teams with no points (due to withdrew).</p><br />
<p><br />
Correlations studies between this number of points and other variables show that that for all teams, the main variables explaining success in iGEM is the number of years of existence and the size of the team. It would therefore seem that mixity would not be a factor. However, when looking at correlations between variables of teams who truly succeeded (points > 20) , the variables that have a significant correlation with the number of points become the sex ratio and the number of supervisors. Therefore it could be hypothesized that beginning iGEM teams have to face major challenges but when the team existed for a few years and general organization or funding problems have been dealt with , diversity could be a factor for success. </p><br />
<p><br />
In order to check if this could be seen in the best iGEM teams that existed, the sex ratio of of prize winner teams was compared to the one of participating teams with boostrap resampling giving a p-value of 0.035 This means that the sex ratio of winning teams (45%) is significantly different from the one of participating teams (37%)<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png" width="400px" /><br />
<b>Figure 8. </b> Gender balance and succes in iGEM<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Clues"></div><br />
<h2> Clues to improve mixity </h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
<br />
Women are not as represented as men in iGEM. Why should this be a problem ? Indeed, even if it might lead to success as explained above, the need to have gender equality could be questioned. However iGEM is an international competition. One of its main goals is to attract and educate young people as well as trying to have them solve real issues. Synthetic biology might be a key technology to solve the main challenges of the 21st century.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p> The world will need science and if iGEM only succeeds in motivating half of the population that could be interested, this would be a major failure to achieve its mission. Therefore, the last part of the study was aimed at understanding how could iGEM improve mixity within its own ranks.<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div class="leftparagraph"><br />
<h3> From the data </h3><br />
<p> &nbsp;&nbsp;<br />
By looking at correlation between sex ratios and other variables, the most striking result is the link between team size and sex ratio. Teams of 2 or 3 people are almost only male teams. Even when taking out those very small teams, out of the data set the correlation holds up. This is a first lead. <br><br />
The second analysis that was made regarding the data was to compare the detailed statistics of the 100 most female teams and 100 male teams. Again, it is found that the total team member is lower for male team (9,7 vs 7,8 (p-value 0,0019) we can hypothesize that having women instructors does matter to attract girls in teams. They serve as role models. Having a woman capable of studying and realizing a synthetic biology project is a direct signal to female students that it is also possible for them to do it. Having a woman adviser might also help girls better adapt in a group and reduce their fears about having to endure constant teasing or "male " ambiance.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<h3> From a survey</h3><br />
<p> &nbsp;&nbsp;<br />
Finally, a survey was conducted among iGEMers and former iGEMers to understand their motivations and activities in iGEM. The study was designed to be unbiased and to avoid stereotype threat (for example by putting the question about gender in the end among many other pieces of information). It is still available <a href="http://bit.ly/14WykuZ"> here</a>. Participants in the survey had to rank from 1 to 5 (1 being not important, and 5 very important) answers to questions regarding personnal and professional motivations for participating in iGEM as well values and on what did they spend their time. 63 people answered among whom 32% were women.</p><br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/c/c5/PB_GS_Survey1.png" width="500px" /><br />
<b>Figure 9. </b> Results of survey : what did you hope to learn in iGEM ?<br />
</center><br />
<br><br><br />
<p><br />
It is interesting to notice that men and women answered almost exactly the same way regarding most of the questions. Women gave a little more importance for the value of fundamental research in iGEM while men graded a bit better "Changing the world". Motivations were approximately the same as well as time spent on each activity. Just a little fact was that men considered human practices a bit more important than women did but spent a little less time on it. <br />
There is only one main difference (more than one point out of five which is represented below) : the will to lead a project and lead a team. It is striking to see how much men are more motivated to lead teams than women. This is definitely to put in relation with the number of women advisers found and the impact it can then have on teams mixity. This could reflect women lack of self esteem in some parts of their work.</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Recommendations"></div><br />
<h2> Recommendations</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Considering all the results that were presented above, here is a list of recommendations for the iGEM foundation to pursue an active policy to improve mixity in iGEM.<br />
<ul><br />
<li> Raise the number of women judges </li> <br />
<li> Promote large teams </li> <br />
<li> Write up a small paragraph to team heads to insist on the importance of motivating young women to be advisers.</li><br />
<li> Giving Bonus point when the team have women advisers </li><br />
</ul><br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<p>And finally, add in iGEM requirements a Gender reflection. By having teams filling out the database that was built and answering the survey and write a small paragraph about how they see mixity in their team and what it could bring, it would drastically raise the awareness of young men and women about the gender problem in science. Having an up-to-date database is also a great way to see improvements in a quantitative manner. It would allow a direct assessment of the effects of an active gender policy which would be a unique example in science. iGEM could become a leader in that fight and prepare the new generation of scientists to finally get rid of the gender inequality in science<br />
</p><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Litterature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>P. Allotey, M. Gyapong Gender in tuberculosis research INT J TUBERC LUNG DIS 2008 </li><br />
<li>M. Calid, S. Rasul, S Ullah Khan, M; Saeed Gender differences in delay to s to tuberculosis diagnosis and treatment outcome<br><br />
European Commission She figures 2012<br><br />
European Commission, Mapping the gaze : getting more women to the top in Research 2008.<br><br />
</li><br />
<li>C.B. Holmes, H. Hausler, P. Hunn : A review of sex differences in the epidemiology of tuberculosis</li><br />
<li>A. N. Martinez J. T. Rhee, P. M. Small,‡M. A. Behr Sex differences in the epidemiology of tuberculosis in San Francisco INT J TUBERC LUNG DIS 4(1):26–31 2000</li><br />
<li>Moss-Racusin et al, (2012) Science faculty’s subtle gender biases favor male students PNAS </li><br />
<br />
</ul><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>McKinsey and Company Women Matter, 2007 </li><br />
<li>Olivier Neyrolles, Lluis Quintana-Murci Sexual Inequality in Tuberculosis, Plos Medicine 2009</li><br />
<li>Nosek et al. (2009) National differences in gender–science stereotypes predict national sex differences in science and math achievement. PNAS June 30, 2009 vol. 106 no. 26 10593–10597</li><br />
<br />
<li>E. Pollack Why Are There Still So Few Women in Science? NY TImes OCtober 2013</li><br />
<li>Al S. Rhines The role of sex differences in the prevalence and transmission of tuberculosis : Tuberculosis 2013</li><br />
<li>M. W. Uplekar, S. Rangan, M. G. Weiss, J. Ogden, M. W. Borgdorff, P. Hudelson Attention to gender issues in tuberculosis control INT J TUBERC LUNG DIS 4(1):26–31 2001</li><br />
</ul><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Attributions</h2><br />
<p>We would like to thank Flora Vincent, President of <a href="http://wax-science.fr/")>WAX Science</a> association for her precious help in analyzing the results, and Kim de Mora and Kitwa from the iGEM foundation, for helping spreading the survey. <p><br />
<p>This project was designed and accomplished by Aude Bernheim, Clovis Basier, Matt Deyell, Marguerite Benony and Sebastian Jaramillo in consultation with Edwin Wintermute and Ariel Lindner.</p><br />
</div><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_StudyTeam:Paris Bettencourt/Human Practice/Gender Study2013-10-28T22:28:53Z<p>Yzegman: </p>
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<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Science suffers from gender bias</p><br />
</div><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Revealed gender bias in synthetic biology by studying sex ratios in SB conferences and labs</li><br />
<li>Built a database of all iGEM teams reporting all available online information and sex ratios of teams and advisors</li><br />
<li>Conducted a statistical analysis of this data-set and showed among other results that success in iGEM is correlated to gender mix</li><br />
<li>Made recommendations to implement an active gender policy in iGEM</li><br />
</ul><br />
<p></p><br />
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<div class="aims"><br />
<h2>Aims</h2><br />
<p>To investigate gender dynamics in iGEM and in synthetic biology research community at large in a quantitative manner</p><br />
</div><br />
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<div style="clear: both;"></div> <br />
<div style="height:68px"> <br />
<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
</div><br />
</a><br />
<a href="#Recommendations"><br />
<div class="hlink"><br />
<h2>Skip to Recommendations</h2><br />
</div><br />
</a><br />
<a href="#Database"><br />
<div class="hlink"><br />
<h2>Skip to Database</h2><br />
</div><br />
</a><br />
<a href="#Findings"> <br />
<div class="hlink" style="margin-right:0px"><br />
<h2>Skip to Main Findings</h2><br />
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<a href="#SynBio"><br />
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<h2>Skip to Gender Bias in SynBio</h2><br />
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</a><br />
<a href="#Success"><br />
<div class="hlink" style="width:355px"><br />
<h2>Skip to iGEM Diversity and Success</h2><br />
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</a><br />
<a href="#Clues"><br />
<div class="hlink" style="width:355px;margin-right:0"><br />
<h2>Skip to Clues to Improve Balance</h2><br />
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<a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Facts"><br />
<div class="hlink" style="width:100%;margin-right:0"><br />
<h2 style="font-size:22px;">Infographics on gender and Synthetic Biology</h2><br />
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<br />
<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
For every woman killed by TB, there are two men. Our review of the literature on gender bias and tuberculosis can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study/Gender_Bias">here</a>. If a disease can be biased, what about ourselves? iGEM? Synthetic biology? Gender bias in science may appear in different forms. Gender balance varies by discipline, by job title, by age or by region. Only 30% of researchers in Europe are women, while 92% of French university deans are men. </p> <p>Hisorically, gender bias has affected the lives of scientists and the practice of science. However, assessing gender bias today in a living community is very difficult. History, stereotypes, limits of the disciplines, and the simple lack of data can prevent us, the synthetic biologists, from thinking about our own relationship to gender.</p><br />
<br><br />
</div><br />
<div class="rightparagraph"><br />
<p><br />
Most of those issues should not apply in synthetic biology. Synthetic biology is a new field. The argument of the heritage of some habits cannot be made. It is a mix of previously existing disciplines and therefore very open and should not reflect preexisting stereotypes. To study gender bias in iGEM and in synthetic biology we decided to follow a data driven approach. Studying in a quantitative manner this subjects had two main benefits. First it prevented us to apply our own biases and stereotypes on this subject. Secondly, it lead us to construct data base that we make freely available and let anyone test his own hypothesis on this controversial subject and form his own conclusions.</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="SynBio"></div><br />
<h2> Synthetic biology field : general overview of gender equality in synthetic biology </h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Gender repartition in synthetic biology can be looked at from different perspectives. For this study, two main ways were chosen: composition of labs and conferences. The main reasons for those choices were the accessibility of online data </p><br />
</div><br />
<div class="rightparagraph"><br />
<p><br />
as well as the necessity to get information not only about the general gender balance but also the sex ratio inside a defined category: PhD students, post docs, head of labs... </p><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2> Synthetic biology labs, a good representation of gender (in)equality in science </h2> <br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Teams of 50 synthetic biology labs were studied. The labs were chosen by their presence on the webpage http://syntheticbiology.org/Labs.html . For each lab, several numbers were reported in a table : total number of people in the team, number of women in the team, number of PhD students, post docs, head of labs, number of women PhD students, post docs, head of labs. From this, the sex ratios (number of women / total number of people) were then calculated for each of those categories. </p><br />
<p><center><a href="https://2013.igem.org/File:ParisB2013_Synthetic_Biology_Research_Groups.xls">Download the database here</a></center></p> <br />
<p><br />
The first conclusion that can be made is that women are generally under-represented in synthetic biology labs. 33% correspond to the average presence of women in research in Europe. Indeed according to the European Commission, 32% of researchers in Europe are women <i>(She Figures, 2012)</i>. <br><br>The second finding also reflects well an already known reality in science : the glass ceiling. In 1995, the glass ceiling was defined by the U.S. Department of Labor, as a <i>"political term used to describe "the unseen, yet unbreakable barrier that keeps minorities and women from rising to the upper rungs of the corporate ladder, regardless of their qualifications or achievements" </i>.<br />
With only 17,85\% of heads of labs being women, synthetic biology is still doing slightly better than the average. According to a European study done in 2008 called <i>Mapping the maze,getting women to the top in research</i>., only 15% of women occupy top research position in Europe. However, the number of SB P.I. should be analyzed through the filter of history. In a new field, it would be expected in a world where bias would not be present anymore to have way more women at those positions.<br />
</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
</br></br></br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/4/46/PB_GS_LabsBis.png" width="353px"/><br><br />
<b>Figure 1. </b> : Sex ratio in synthetic biology labs<br />
</center><br />
<br />
</br></br></br><br />
<center><br />
<TABLE BORDER="1"> <br />
<CAPTION> </CAPTION> <br />
<TR> <br />
<TH>Labs </TH> <br />
<TH> Phd Students </TH> <br />
<TH> Post Docs</TH> <br />
<TH> Head of Labs </TH> <br />
</TR> <br />
<TR> <br />
<TH> 33,10 % </TH> <br />
<TD> 35,39 % </TD> <br />
<TD> 31,31 % </TD> <br />
<TD> 17,85 % </TD> <br />
</TR> <br />
</TABLE><br />
</center><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2> Speakers at SB Conferences : effects of an active gender policy</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SBX.0 conferences accompanied the development of synthetic biology. They provide a great way to investigate the evolution of gender ratio since the birth of synthetic biology. Moreover, the presence/absence of women as speakers is a known indicator of gender bias and specially of active gender policy. Indeed, several social mechanisms are in place lead to fewer female speakers that could be expected: self censorship, unconscious stereotypes, unconscious choice of only male speakers... However, having female speakers at conference is a key point. It allows women, to gain confidence but also to act as role model for women attending the conference. </p><br />
<p><br />
To study SB conferences, available programs online were downloaded. Data referring to the number of speakers but also to posters were recorded. The data-set could not be completed for certain years due to the impossibility of finding the data online.<br />
</p><br />
<p><center><a href="https://2013.igem.org/File:ParisB2013Resultats_SB.xls">Download the database here</a></center></p><br />
<p><br />
The sex ratio of the speakers have followed a very interesting evolution. It has been multiplied by 3 from SB1 to SB5. This could indicate a change of policy considering speakers. Most likely, the first conferences invited speakers without taking into consideration the gender dimension. Might it be due to some complaints or the raise in awareness of the conferences organizers, the numbers went up. This example is interesting because it clearly show an interest in the subject by the involved community.<br />
</p><br />
<p><br />
Two main conclusions can be drawn on posters. First, the sex ratio of authors in posters has changed throughout the years. Secondly, this number is not as high as the sex ratio in labs. The question is why? The points described above could be underlying reasons, however it is very difficult to truly go beyond this with only those numbers. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<br><br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/6/62/PB_GS_Sex_ratio_in_SB_Conf.png" width="400px"/><br><br />
<b> Figure 2. </b> Sex ratio in SB conferences<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2> Under represented and badly represented</h2> <br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
In order to try to better understand the dynamics of gender behind the posters numbers, the rank of authors were reported for each poster. Sex ratio were calculated for each rank, keeping in mind that in biology, the first author is often a Phd student or a post doc and the last author, the P.I.<br />
</p><br />
<p><br />
As explained above, women are generally under-represented in synthetic biology labs, even less represented at conferences. When looking at the rank of author in posters, another bias appears. Indeed, women are more likely to be present as middle authors than first or last. This bias can be found in papers of different disciplines as shown on the graph realized on the eigenfactor.</p><br />
<p> <br />
The main finding considering gender in synthetic biology is that even though synthetic biology is new and interdisciplinary, it remains quite representative of existing gender bias in science. Therefore it can be concluded, that the issues that have kept women out of science and especially out of top research position are still present and will not be resolved with time. A strong and active policy appears necessary to bring more mixity and therefore diversity in this field.<br />
<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/PB_GS_Author_place_.png" width="435px"/> <br><br />
<b>Figure 3. </b> Sex ratio according to rank of authors in SB posters.<br />
</center><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/PB_GS_Eigenfactor.png" width="535px"/><br />
<b>Figure 4. </b> : Analysis of rank of authors according to gender in scientific publications<br />
</center> <br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Database"></div><br />
<h2> iGEM as a model : a fantastic database </h2><br />
<div class="leftparagraph"><br />
<h3> Online Data</h3> <br />
<p> &nbsp;&nbsp;<br />
All the data concerning iGEM were retrieved from the website : <a href="https://igem.org">https://igem.org</a> <br />
List of teams were retrieved from the webpages <a href="https://igem.org/Team_List.cgi?year=2012">https://igem.org/Team_List.cgi?year=2012</a>.<br />
List of project themes were retrieved from <a href="https://igem.org/Team_Tracks?year=2012">https://igem.org/Team_Tracks?year=2012</a>.<br />
List of prices were retrieved <a href="https://igem.org/Results">https://igem.org/Results</a>.<br />
List of judges were retrieved from: <a href="https://igem.org/Judge_List">https://igem.org/Judge_List</a><br />
</p><br />
<h3> Sex ratio determination :</h3> <br />
<p> &nbsp;&nbsp; For each team, the official team profile was checked to count the number of student members, advisors and instructors.<br />
Then to determine the sex of particpants, wiki were used when names were not obvious, using pictures when they existed. When no pictures were available and names were not obviously referring to one sex, a google image search was done on the name (first and last name) and the sex was chosen as the most represented sex in the pictures (if 10 images of men come up and 30 of women, the participant was considered as a woman).</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<h3> Database : </h3> <br />
<p> &nbsp;&nbsp; Information for the first year of iGEM were difficult to find because of the non existence of available wiki pages and it was therefore decided not to take into account this year.<br />
Teams who withdrew during the competition were not taken into account since it was most of the time impossible to know the number of participants due to the absence of wiki.<br />
In the end our data set is composed of 662 teams over 5 years. For each team were reported : <br />
Year ; region ; name of the team ; number of student members ; number of women student members ; number of advisors ; number of women advisors ; number of instructors ; number of women instructors ; participation to MIT championship ; medal ; regional prices ; championship prices ;tracks. </p><br />
<p><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2013/3/35/PB_GS_IGEMdatabase.xls"> Download the database here</a><br />
</center><br />
</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Findings"></div><br />
<h2> iGEM : a mirror of main gender problems </h2><br />
<div class="leftparagraph"><br />
<h3> Teams sex ratio, a very robust value </h3> <br />
<p> &nbsp;&nbsp;<br />
<br><br><br />
The first thing that was examined was the evolution of sex ratio of teams in iGEM across continents and throughout the years.<br />
</p><br />
<br><br />
<p><br />
The striking conclusion of this comparison is that the sex ratio is iGEM teams remains constant through the years and across continents (ANOVA's p-value for the different conditions > 0,5). This shows that women are underrepresented in iGEM teams. </p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/5/5a/GS_Year.png" width="250px"/><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/GS_Region.png" width="250px"/><br />
<b>Figure 5. </b> Sex ratio of iGEM teams through the years and across continents<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="leftparagraph"><br />
<h3> Women do not supervise as much as men </h3> <br />
<p> &nbsp;&nbsp;<br />
The second question investigated was the sex ratios for the different categories of people participating in iGEM. Indeed, iGEM is not only undergrad students. Advisors, instructors, judges also participate representing the complete professional ladder of synthetic biology. A category called Supervisors was created corresponding to instructors and advisers. Indeed, those terms are not understood and used in the same way in different continents. In some countries "advisers" means people who directly teach the teams (mostly grad students and post docs) whereas it means general mentors for others and vice versa.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/c/c6/PB_GS_Sex_ratio_in_iGEM_according_to_role.png" width="300px" height="250px"/> <br><br />
<b>Figure 6. </b> Sex ratios in iGEM according to categories of people participating<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
When executing comparisons tests , team members' sex ratio is found to be different from judges' and instructors' ones (p value < 0,01). However judges and advisers are not significantly different ( p value > 0,5). This result reveals a tendency of women to supervise less than men. Indeed, from team members to instructors, the sex ratio is divided by two. What is even more interesting is to compare those numbers to sex ratios of PhD students and post docs in labs. The sex ratio of instructors is 10 points lower. </p><br />
<p> Women constitute a pool of talent that is not mobilized. They participate but do not supervise teams. They are "lost" along the way. Indeed, in a study published last year in PNAS, researchers showed that P.I. were less prone to have a woman mentoring students than man. This unconscious bias can be translated by a lack of encouragement from P.I.s but also by a self censorship which is not taken into account by other supervisors as explained in an recently published article by Eileen Pollack (E. Pollack Why Are There Still So Few Women in Science? NY TImes OCtober 2013).<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<h3> Tracks and sex ratio in iGEM </h3> <br />
<p> &nbsp;&nbsp;<br />
The third finding goes against an often-heard stereotype "women are more interested by applied research". In order to investigate this subject, tracks were reported for each project. In iGEM tracks correspond to general theme of the project : medicine, fundationnal research…Tracks were then looked at in terms of sex ratios. There is no significant difference between tracks. (ANOVA > 0,1).</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/PB_GS_Sexratiotracksbis.png" width="535px"/><br />
<b>Figure 7. </b> Sex ratios and tracks in iGEM<br />
</center><br />
<p><br />
To conclude, studying the iGEM competition gives a unique quantitative insight on existing questions in the field of gender studies. It also constitutes an amazing argument to convince scientists of the existence of a gender issue in science. As explained by Rascun et al in a recent paper published in PNAS, scientists believe that those type of bias only exist in some labs, not their own, therefore very objective studies need to be conducted to clearly show the reality of the numbers. More over , Jo Handelsman a microbiologist involved in that paper underlined in a recent interview, that people often think that there is still an issue in physics or maths but that there are no more women issues in biology, which is not true. This study supports strongly the view that this general thinking is untrue.<br />
</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Success"></div><br />
<h3> In iGEM, is diversity a factor of success ? </h3><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Several studies led by consulting groups (McKinsey and Company Women Matter, 2007) have shown that mixity in a team increases performance. The big question of what leads to success in iGEM was therefore investigated using the database with a special focus on gender. In order to be able to get a general idea about iGEM team success, a point system was put in place.<br />
</p><br />
<p>Points were attributed the following way.<br><br />
For the medal: 1 point for bronze medal, 2 points for silver medal, 3 points for gold medal. For the world jamboree qualified teams: 2 points for every team taking part in 2010 and before (before regional jamborees existed) , 6 points for team qualified for world final (after 2010). For special prices (Best ...): 6 points were attributed for each regional price earned (only after 2010), 13 points for each price earned in the world final (all price worth 13 points before regional jamborees existed). For the final place in world final: 15 points for the sixth team, 20 points for the fifth team, 25 points for the fourth team, 30 points for the third team, 35 points for the second team, 40 points for the firth team.</p><br />
<p><br />
The aim was to give each team a score that is proportional to the rewards it earned, taking in account that all teams were in world jamboree prior to 2011, without having to be qualified in regional jamborees.<br />
</p><br />
<p>Best score is for the Imperial College London team in 2011 (81 points).<br />
All teams (all years) average is 7.41 points, considering teams with no points (due to withdrew).</p><br />
<p><br />
Correlations studies between this number of points and other variables show that that for all teams, the main variables explaining success in iGEM is the number of years of existence and the size of the team. It would therefore seem that mixity would not be a factor. However, when looking at correlations between variables of teams who truly succeeded (points > 20) , the variables that have a significant correlation with the number of points become the sex ratio and the number of supervisors. Therefore it could be hypothesized that beginning iGEM teams have to face major challenges but when the team existed for a few years and general organization or funding problems have been dealt with , diversity could be a factor for success. </p><br />
<p><br />
In order to check if this could be seen in the best iGEM teams that existed, the sex ratio of of prize winner teams was compared to the one of participating teams with boostrap resampling giving a p-value of 0.035 This means that the sex ratio of winning teams (45%) is significantly different from the one of participating teams (37%)<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png" width="400px" /><br />
<b>Figure 8. </b> Gender balance and succes in iGEM<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Clues"></div><br />
<h2> Clues to improve mixity </h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
<br />
Women are not as represented as men in iGEM. Why should this be a problem ? Indeed, even if it might lead to success as explained above, the need to have gender equality could be questioned. However iGEM is an international competition. One of its main goals is to attract and educate young people as well as trying to have them solve real issues. Synthetic biology might be a key technology to solve the main challenges of the 21st century.</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<p> The world will need science and if iGEM only succeeds in motivating half of the population that could be interested, this would be a major failure to achieve its mission. Therefore, the last part of the study was aimed at understanding how could iGEM improve mixity within its own ranks.<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<div class="leftparagraph"><br />
<h3> From the data </h3><br />
<p> &nbsp;&nbsp;<br />
By looking at correlation between sex ratios and other variables, the most striking result is the link between team size and sex ratio. Teams of 2 or 3 people are almost only male teams. Even when taking out those very small teams, out of the data set the correlation holds up. This is a first lead. <br><br />
The second analysis that was made regarding the data was to compare the detailed statistics of the 100 most female teams and 100 male teams. Again, it is found that the total team member is lower for male team (9,7 vs 7,8 (p-value 0,0019) we can hypothesize that having women instructors does matter to attract girls in teams. They serve as role models. Having a woman capable of studying and realizing a synthetic biology project is a direct signal to female students that it is also possible for them to do it. Having a woman adviser might also help girls better adapt in a group and reduce their fears about having to endure constant teasing or "male " ambiance.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<h3> From a survey</h3><br />
<p> &nbsp;&nbsp;<br />
Finally, a survey was conducted among iGEMers and former iGEMers to understand their motivations and activities in iGEM. The study was designed to be unbiased and to avoid stereotype threat (for example by putting the question about gender in the end among many other pieces of information). It is still available <a href="http://bit.ly/14WykuZ"> here</a>. Participants in the survey had to rank from 1 to 5 (1 being not important, and 5 very important) answers to questions regarding personnal and professional motivations for participating in iGEM as well values and on what did they spend their time. 63 people answered among whom 32% were women.</p><br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/c/c5/PB_GS_Survey1.png" width="500px" /><br />
<b>Figure 9. </b> Results of survey : what did you hope to learn in iGEM ?<br />
</center><br />
<br><br><br />
<p><br />
It is interesting to notice that men and women answered almost exactly the same way regarding most of the questions. Women gave a little more importance for the value of fundamental research in iGEM while men graded a bit better "Changing the world". Motivations were approximately the same as well as time spent on each activity. Just a little fact was that men considered human practices a bit more important than women did but spent a little less time on it. <br />
There is only one main difference (more than one point out of five which is represented below) : the will to lead a project and lead a team. It is striking to see how much men are more motivated to lead teams than women. This is definitely to put in relation with the number of women advisers found and the impact it can then have on teams mixity. This could reflect women lack of self esteem in some parts of their work.</p><br />
</div><br />
<div style="clear: both;"></div><br />
<div id="Recommendations"></div><br />
<h2> Recommendations</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Considering all the results that were presented above, here is a list of recommendations for the iGEM foundation to pursue an active policy to improve mixity in iGEM.<br />
<ul><br />
<li> Raise the number of women judges </li> <br />
<li> Promote large teams </li> <br />
<li> Write up a small paragraph to team heads to insist on the importance of motivating young women to be advisers.</li><br />
<li> Giving Bonus point when the team have women advisers </li><br />
</ul><br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<p>And finally, add in iGEM requirements a Gender reflection. By having teams filling out the database that was built and answering the survey and write a small paragraph about how they see mixity in their team and what it could bring, it would drastically raise the awareness of young men and women about the gender problem in science. Having an up-to-date database is also a great way to see improvements in a quantitative manner. It would allow a direct assessment of the effects of an active gender policy which would be a unique example in science. iGEM could become a leader in that fight and prepare the new generation of scientists to finally get rid of the gender inequality in science<br />
</p><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Litterature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>P. Allotey, M. Gyapong Gender in tuberculosis research INT J TUBERC LUNG DIS 2008 </li><br />
<li>M. Calid, S. Rasul, S Ullah Khan, M; Saeed Gender differences in delay to s to tuberculosis diagnosis and treatment outcome<br><br />
European Commission She figures 2012<br><br />
European Commission, Mapping the gaze : getting more women to the top in Research 2008.<br><br />
</li><br />
<li>C.B. Holmes, H. Hausler, P. Hunn : A review of sex differences in the epidemiology of tuberculosis</li><br />
<li>A. N. Martinez J. T. Rhee, P. M. Small,‡M. A. Behr Sex differences in the epidemiology of tuberculosis in San Francisco INT J TUBERC LUNG DIS 4(1):26–31 2000</li><br />
<li>Moss-Racusin et al, (2012) Science faculty’s subtle gender biases favor male students PNAS </li><br />
<br />
</ul><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>McKinsey and Company Women Matter, 2007 </li><br />
<li>Olivier Neyrolles, Lluis Quintana-Murci Sexual Inequality in Tuberculosis, Plos Medicine 2009</li><br />
<li>Nosek et al. (2009) National differences in gender–science stereotypes predict national sex differences in science and math achievement. PNAS June 30, 2009 vol. 106 no. 26 10593–10597</li><br />
<br />
<li>E. Pollack Why Are There Still So Few Women in Science? NY TImes OCtober 2013</li><br />
<li>Al S. Rhines The role of sex differences in the prevalence and transmission of tuberculosis : Tuberculosis 2013</li><br />
<li>M. W. Uplekar, S. Rangan, M. G. Weiss, J. Ogden, M. W. Borgdorff, P. Hudelson Attention to gender issues in tuberculosis control INT J TUBERC LUNG DIS 4(1):26–31 2001</li><br />
</ul><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Attributions</h2><br />
<p>We would like to thank Flora Vincent, President of <a href="http://wax-science.fr/")>WAX Science</a> association for her precious help in analyzing the results, and Kim de Mora and Kitwa from the iGEM foundation, for helping spreading the survey. <p><br />
<p>This project was designed and accomplished by Aude Bernheim, Clovis Basier, Matt Deyell, Marguerite Benony and Sebastian Jaramillo in consultation with Edwin Wintermute and Ariel Lindner.</p><br />
</div><br />
</div><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/File:GS_Year.pngFile:GS Year.png2013-10-28T22:25:51Z<p>Yzegman: </p>
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<div></div>Yzegmanhttp://2013.igem.org/File:Newfiguresabotagepresentation.pngFile:Newfiguresabotagepresentation.png2013-10-28T22:23:35Z<p>Yzegman: uploaded a new version of &quot;File:Newfiguresabotagepresentation.png&quot;</p>
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<div></div>Yzegmanhttp://2013.igem.org/File:Newfiguresabotagepresentation.pngFile:Newfiguresabotagepresentation.png2013-10-28T22:23:12Z<p>Yzegman: uploaded a new version of &quot;File:Newfiguresabotagepresentation.png&quot;</p>
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<div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:17:31Z<p>Yzegman: </p>
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<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
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<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<style><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
<style><br />
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<div class="overbox"><br />
<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender in(equality) in iGEM and synthetic biology. A database was gathered and statistically analysed in order to depict sex ratio of iGEM teams' students and supervisors. <br />
<br><center><img width="50%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:14:34Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
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<div id="grouptitle"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<style><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
<style><br />
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<div class="overbox"><br />
<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender in(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<br><center><img width="50%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:13:57Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
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<div id="grouptitle"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<style><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender in(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<br><img width="50%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:13:17Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
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<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<br><img width="50%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:12:49Z<p>Yzegman: </p>
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<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
<style><br />
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<div class="overbox"><br />
<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<br><img width="10%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:11:41Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
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<div id="grouptitle"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<style><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
<style><br />
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<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<img height="10%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:11:14Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
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<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<style><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<div class="overbox"><br />
<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<<img height="30%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:09:57Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
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<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<br><img style="height=30%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:09:35Z<p>Yzegman: </p>
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{{:Team:Paris_Bettencourt/Menu}}<br />
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<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<style><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
<style><br />
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<div class="overbox"><br />
<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<br><img src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:08:49Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
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<div id="grouptitle"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<style><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
<style><br />
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<div class="overbox"><br />
<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<br><center><img style="height=30%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T22:08:29Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
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<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<style><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
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<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<br><center><img style="height=50%" src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
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<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
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<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_StudyTeam:Paris Bettencourt/Human Practice/Gender Study2013-10-28T21:49:43Z<p>Yzegman: </p>
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<h2>Background</h2><br />
<p>Science suffers from gender bias</p><br />
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<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Revealed gender bias in synthetic biology by studying sex ratios in SB conferences and labs</li><br />
<li>Built a database of all iGEM teams reporting all available online information and sex ratios of teams and advisors</li><br />
<li>Conducted a statistical analysis of this data-set and showed among other results that success in iGEM is correlated to gender mix</li><br />
<li>Made recommendations to implement an active gender policy in iGEM</li><br />
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<div class="aims"><br />
<h2>Aims</h2><br />
<p>To investigate gender dynamics in iGEM and in synthetic biology research community at large in a quantitative manner</p><br />
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<a href="#Introduction"><br />
<div class="hlink"><br />
<h2>Skip to Introduction</h2><br />
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<h2>Skip to Recommendations</h2><br />
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<h2>Skip to Database</h2><br />
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<a href="#Findings"> <br />
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<h2>Skip to Main Findings</h2><br />
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<h2>Skip to Gender Bias in SynBio</h2><br />
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<h2>Skip to iGEM Diversity and Success</h2><br />
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<a href="#Clues"><br />
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<h2>Skip to Clues to Improve Balance</h2><br />
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<a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Facts"><br />
<div class="hlink" style="width:100%;margin-right:0"><br />
<h2 style="font-size:22px;">Infographics on gender and Synthetic Biology</h2><br />
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<div id="Introduction"></div><br />
<h2>Introduction</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
For every woman killed by TB, there are two men. Our review of the literature on gender bias and tuberculosis can be found <a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study/Gender_Bias">here</a>. If a disease can be biased, what about ourselves? iGEM? Synthetic biology? Gender bias in science may appear in different forms. Gender balance varies by discipline, by job title, by age or by region. Only 30% of researchers in Europe are women, while 92% of French university deans are men. </p> <p>Hisorically, gender bias has affected the lives of scientists and the practice of science. However, assessing gender bias today in a living community is very difficult. History, stereotypes, limits of the disciplines, and the simple lack of data can prevent us, the synthetic biologists, from thinking about our own relationship to gender.</p><br />
<br><br />
</div><br />
<div class="rightparagraph"><br />
<p><br />
Most of those issues should not apply in synthetic biology. Synthetic biology is a new field. The argument of the heritage of some habits cannot be made. It is a mix of previously existing disciplines and therefore very open and should not reflect preexisting stereotypes. To study gender bias in iGEM and in synthetic biology we decided to follow a data driven approach. Studying in a quantitative manner this subjects had two main benefits. First it prevented us to apply our own biases and stereotypes on this subject. Secondly, it lead us to construct data base that we make freely available and let anyone test his own hypothesis on this controversial subject and form his own conclusions.</p><br />
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<div id="SynBio"></div><br />
<h2> Synthetic biology field : general overview of gender equality in synthetic biology </h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Gender repartition in synthetic biology can be looked at from different perspectives. For this study, two main ways were chosen: composition of labs and conferences. The main reasons for those choices were the accessibility of online data </p><br />
</div><br />
<div class="rightparagraph"><br />
<p><br />
as well as the necessity to get information not only about the general gender balance but also the sex ratio inside a defined category: PhD students, post docs, head of labs... </p><br />
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<br />
<h2> Synthetic biology labs, a good representation of gender (in)equality in science </h2> <br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Teams of 50 synthetic biology labs were studied. The labs were chosen by their presence on the webpage http://syntheticbiology.org/Labs.html . For each lab, several numbers were reported in a table : total number of people in the team, number of women in the team, number of PhD students, post docs, head of labs, number of women PhD students, post docs, head of labs. From this, the sex ratios (number of women / total number of people) were then calculated for each of those categories. </p><br />
<p><center><a href="https://2013.igem.org/File:ParisB2013_Synthetic_Biology_Research_Groups.xls">Download the database here</a></center></p> <br />
<p><br />
The first conclusion that can be made is that women are generally under-represented in synthetic biology labs. 33% correspond to the average presence of women in research in Europe. Indeed according to the European Commission, 32% of researchers in Europe are women <i>(She Figures, 2012)</i>. <br><br>The second finding also reflects well an already known reality in science : the glass ceiling. In 1995, the glass ceiling was defined by the U.S. Department of Labor, as a <i>"political term used to describe "the unseen, yet unbreakable barrier that keeps minorities and women from rising to the upper rungs of the corporate ladder, regardless of their qualifications or achievements" </i>.<br />
With only 17,85\% of heads of labs being women, synthetic biology is still doing slightly better than the average. According to a European study done in 2008 called <i>Mapping the maze,getting women to the top in research</i>., only 15% of women occupy top research position in Europe. However, the number of SB P.I. should be analyzed through the filter of history. In a new field, it would be expected in a world where bias would not be present anymore to have way more women at those positions.<br />
</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
</br></br></br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/4/46/PB_GS_LabsBis.png" width="353px"/><br><br />
<b>Figure 1. </b> : Sex ratio in synthetic biology labs<br />
</center><br />
<br />
</br></br></br><br />
<center><br />
<TABLE BORDER="1"> <br />
<CAPTION> </CAPTION> <br />
<TR> <br />
<TH>Labs </TH> <br />
<TH> Phd Students </TH> <br />
<TH> Post Docs</TH> <br />
<TH> Head of Labs </TH> <br />
</TR> <br />
<TR> <br />
<TH> 33,10 % </TH> <br />
<TD> 35,39 % </TD> <br />
<TD> 31,31 % </TD> <br />
<TD> 17,85 % </TD> <br />
</TR> <br />
</TABLE><br />
</center><br />
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<br />
<h2> Speakers at SB Conferences : effects of an active gender policy</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
SBX.0 conferences accompanied the development of synthetic biology. They provide a great way to investigate the evolution of gender ratio since the birth of synthetic biology. Moreover, the presence/absence of women as speakers is a known indicator of gender bias and specially of active gender policy. Indeed, several social mechanisms are in place lead to fewer female speakers that could be expected: self censorship, unconscious stereotypes, unconscious choice of only male speakers... However, having female speakers at conference is a key point. It allows women, to gain confidence but also to act as role model for women attending the conference. </p><br />
<p><br />
To study SB conferences, available programs online were downloaded. Data referring to the number of speakers but also to posters were recorded. The data-set could not be completed for certain years due to the impossibility of finding the data online.<br />
</p><br />
<p><center><a href="https://2013.igem.org/File:ParisB2013Resultats_SB.xls">Download the database here</a></center></p><br />
<p><br />
The sex ratio of the speakers have followed a very interesting evolution. It has been multiplied by 3 from SB1 to SB5. This could indicate a change of policy considering speakers. Most likely, the first conferences invited speakers without taking into consideration the gender dimension. Might it be due to some complaints or the raise in awareness of the conferences organizers, the numbers went up. This example is interesting because it clearly show an interest in the subject by the involved community.<br />
</p><br />
<p><br />
Two main conclusions can be drawn on posters. First, the sex ratio of authors in posters has changed throughout the years. Secondly, this number is not as high as the sex ratio in labs. The question is why? The points described above could be underlying reasons, however it is very difficult to truly go beyond this with only those numbers. <br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<br><br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/6/62/PB_GS_Sex_ratio_in_SB_Conf.png" width="400px"/><br><br />
<b> Figure 2. </b> Sex ratio in SB conferences<br />
</center><br />
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<br />
<h2> Under represented and badly represented</h2> <br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
In order to try to better understand the dynamics of gender behind the posters numbers, the rank of authors were reported for each poster. Sex ratio were calculated for each rank, keeping in mind that in biology, the first author is often a Phd student or a post doc and the last author, the P.I.<br />
</p><br />
<p><br />
As explained above, women are generally under-represented in synthetic biology labs, even less represented at conferences. When looking at the rank of author in posters, another bias appears. Indeed, women are more likely to be present as middle authors than first or last. This bias can be found in papers of different disciplines as shown on the graph realized on the eigenfactor.</p><br />
<p> <br />
The main finding considering gender in synthetic biology is that even though synthetic biology is new and interdisciplinary, it remains quite representative of existing gender bias in science. Therefore it can be concluded, that the issues that have kept women out of science and especially out of top research position are still present and will not be resolved with time. A strong and active policy appears necessary to bring more mixity and therefore diversity in this field.<br />
<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/6/6b/PB_GS_Author_place_.png" width="435px"/> <br><br />
<b>Figure 3. </b> Sex ratio according to rank of authors in SB posters.<br />
</center><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/PB_GS_Eigenfactor.png" width="535px"/><br />
<b>Figure 4. </b> : Analysis of rank of authors according to gender in scientific publications<br />
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<div id="Database"></div><br />
<h2> iGEM as a model : a fantastic database </h2><br />
<div class="leftparagraph"><br />
<h3> Online Data</h3> <br />
<p> &nbsp;&nbsp;<br />
All the data concerning iGEM were retrieved from the website : <a href="https://igem.org">https://igem.org</a> <br />
List of teams were retrieved from the webpages <a href="https://igem.org/Team_List.cgi?year=2012">https://igem.org/Team_List.cgi?year=2012</a>.<br />
List of project themes were retrieved from <a href="https://igem.org/Team_Tracks?year=2012">https://igem.org/Team_Tracks?year=2012</a>.<br />
List of prices were retrieved <a href="https://igem.org/Results">https://igem.org/Results</a>.<br />
List of judges were retrieved from: <a href="https://igem.org/Judge_List">https://igem.org/Judge_List</a><br />
</p><br />
<h3> Sex ratio determination :</h3> <br />
<p> &nbsp;&nbsp; For each team, the official team profile was checked to count the number of student members, advisors and instructors.<br />
Then to determine the sex of particpants, wiki were used when names were not obvious, using pictures when they existed. When no pictures were available and names were not obviously referring to one sex, a google image search was done on the name (first and last name) and the sex was chosen as the most represented sex in the pictures (if 10 images of men come up and 30 of women, the participant was considered as a woman).</p><br />
<br />
</div><br />
<div class="rightparagraph"><br />
<h3> Database : </h3> <br />
<p> &nbsp;&nbsp; Information for the first year of iGEM were difficult to find because of the non existence of available wiki pages and it was therefore decided not to take into account this year.<br />
Teams who withdrew during the competition were not taken into account since it was most of the time impossible to know the number of participants due to the absence of wiki.<br />
In the end our data set is composed of 662 teams over 5 years. For each team were reported : <br />
Year ; region ; name of the team ; number of student members ; number of women student members ; number of advisors ; number of women advisors ; number of instructors ; number of women instructors ; participation to MIT championship ; medal ; regional prices ; championship prices ;tracks. </p><br />
<p><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2013/3/35/PB_GS_IGEMdatabase.xls"> Download the database here</a><br />
</center><br />
</p><br />
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<div id="Findings"></div><br />
<h2> iGEM : a mirror of main gender problems </h2><br />
<div class="leftparagraph"><br />
<h3> Teams sex ratio, a very robust value </h3> <br />
<p> &nbsp;&nbsp;<br />
<br><br><br />
The first thing that was examined was the evolution of sex ratio of teams in iGEM across continents and throughout the years.<br />
</p><br />
<br><br />
<p><br />
The striking conclusion of this comparison is that the sex ratio is iGEM teams remains constant through the years and across continents (ANOVA's p-value for the different conditions > 0,5). This shows that women are underrepresented in iGEM teams. </p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/9/9a/PB_GS_Sex_ratio_of_iGEM_teams_per_years.png" width="535px"/><br />
<b>Figure 5. </b> Sex ratio of iGEM teams through the years and across continents<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="leftparagraph"><br />
<h3> Women do not supervise as much as men </h3> <br />
<p> &nbsp;&nbsp;<br />
The second question investigated was the sex ratios for the different categories of people participating in iGEM. Indeed, iGEM is not only undergrad students. Advisors, instructors, judges also participate representing the complete professional ladder of synthetic biology. A category called Supervisors was created corresponding to instructors and advisers. Indeed, those terms are not understood and used in the same way in different continents. In some countries "advisers" means people who directly teach the teams (mostly grad students and post docs) whereas it means general mentors for others and vice versa.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/c/c6/PB_GS_Sex_ratio_in_iGEM_according_to_role.png" width="300px" height="250px"/> <br><br />
<b>Figure 6. </b> Sex ratios in iGEM according to categories of people participating<br />
</center><br />
</div><br />
<div style="clear: both;"></div><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
When executing comparisons tests , team members' sex ratio is found to be different from judges' and instructors' ones (p value < 0,01). However judges and advisers are not significantly different ( p value > 0,5). This result reveals a tendency of women to supervise less than men. Indeed, from team members to instructors, the sex ratio is divided by two. What is even more interesting is to compare those numbers to sex ratios of PhD students and post docs in labs. The sex ratio of instructors is 10 points lower. </p><br />
<p> Women constitute a pool of talent that is not mobilized. They participate but do not supervise teams. They are "lost" along the way. Indeed, in a study published last year in PNAS, researchers showed that P.I. were less prone to have a woman mentoring students than man. This unconscious bias can be translated by a lack of encouragement from P.I.s but also by a self censorship which is not taken into account by other supervisors as explained in an recently published article by Eileen Pollack (E. Pollack Why Are There Still So Few Women in Science? NY TImes OCtober 2013).<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<h3> Tracks and sex ratio in iGEM </h3> <br />
<p> &nbsp;&nbsp;<br />
The third finding goes against an often-heard stereotype "women are more interested by applied research". In order to investigate this subject, tracks were reported for each project. In iGEM tracks correspond to general theme of the project : medicine, fundationnal research…Tracks were then looked at in terms of sex ratios. There is no significant difference between tracks. (ANOVA > 0,1).</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/PB_GS_Sexratiotracksbis.png" width="535px"/><br />
<b>Figure 7. </b> Sex ratios and tracks in iGEM<br />
</center><br />
<p><br />
To conclude, studying the iGEM competition gives a unique quantitative insight on existing questions in the field of gender studies. It also constitutes an amazing argument to convince scientists of the existence of a gender issue in science. As explained by Rascun et al in a recent paper published in PNAS, scientists believe that those type of bias only exist in some labs, not their own, therefore very objective studies need to be conducted to clearly show the reality of the numbers. More over , Jo Handelsman a microbiologist involved in that paper underlined in a recent interview, that people often think that there is still an issue in physics or maths but that there are no more women issues in biology, which is not true. This study supports strongly the view that this general thinking is untrue.<br />
</p><br />
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<div id="Success"></div><br />
<h3> In iGEM, is diversity a factor of success ? </h3><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Several studies led by consulting groups (McKinsey and Company Women Matter, 2007) have shown that mixity in a team increases performance. The big question of what leads to success in iGEM was therefore investigated using the database with a special focus on gender. In order to be able to get a general idea about iGEM team success, a point system was put in place.<br />
</p><br />
<p>Points were attributed the following way.<br><br />
For the medal: 1 point for bronze medal, 2 points for silver medal, 3 points for gold medal. For the world jamboree qualified teams: 2 points for every team taking part in 2010 and before (before regional jamborees existed) , 6 points for team qualified for world final (after 2010). For special prices (Best ...): 6 points were attributed for each regional price earned (only after 2010), 13 points for each price earned in the world final (all price worth 13 points before regional jamborees existed). For the final place in world final: 15 points for the sixth team, 20 points for the fifth team, 25 points for the fourth team, 30 points for the third team, 35 points for the second team, 40 points for the firth team.</p><br />
<p><br />
The aim was to give each team a score that is proportional to the rewards it earned, taking in account that all teams were in world jamboree prior to 2011, without having to be qualified in regional jamborees.<br />
</p><br />
<p>Best score is for the Imperial College London team in 2011 (81 points).<br />
All teams (all years) average is 7.41 points, considering teams with no points (due to withdrew).</p><br />
<p><br />
Correlations studies between this number of points and other variables show that that for all teams, the main variables explaining success in iGEM is the number of years of existence and the size of the team. It would therefore seem that mixity would not be a factor. However, when looking at correlations between variables of teams who truly succeeded (points > 20) , the variables that have a significant correlation with the number of points become the sex ratio and the number of supervisors. Therefore it could be hypothesized that beginning iGEM teams have to face major challenges but when the team existed for a few years and general organization or funding problems have been dealt with , diversity could be a factor for success. </p><br />
<p><br />
In order to check if this could be seen in the best iGEM teams that existed, the sex ratio of of prize winner teams was compared to the one of participating teams with boostrap resampling giving a p-value of 0.035 This means that the sex ratio of winning teams (45%) is significantly different from the one of participating teams (37%)<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png" width="400px" /><br />
<b>Figure 8. </b> Gender balance and succes in iGEM<br />
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<div id="Clues"></div><br />
<h2> Clues to improve mixity </h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
<br />
Women are not as represented as men in iGEM. Why should this be a problem ? Indeed, even if it might lead to success as explained above, the need to have gender equality could be questioned. However iGEM is an international competition. One of its main goals is to attract and educate young people as well as trying to have them solve real issues. Synthetic biology might be a key technology to solve the main challenges of the 21st century.</p><br />
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</div><br />
<div class="rightparagraph"><br />
<p> The world will need science and if iGEM only succeeds in motivating half of the population that could be interested, this would be a major failure to achieve its mission. Therefore, the last part of the study was aimed at understanding how could iGEM improve mixity within its own ranks.<br />
</div><br />
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<br />
<div class="leftparagraph"><br />
<h3> From the data </h3><br />
<p> &nbsp;&nbsp;<br />
By looking at correlation between sex ratios and other variables, the most striking result is the link between team size and sex ratio. Teams of 2 or 3 people are almost only male teams. Even when taking out those very small teams, out of the data set the correlation holds up. This is a first lead. <br><br />
The second analysis that was made regarding the data was to compare the detailed statistics of the 100 most female teams and 100 male teams. Again, it is found that the total team member is lower for male team (9,7 vs 7,8 (p-value 0,0019) we can hypothesize that having women instructors does matter to attract girls in teams. They serve as role models. Having a woman capable of studying and realizing a synthetic biology project is a direct signal to female students that it is also possible for them to do it. Having a woman adviser might also help girls better adapt in a group and reduce their fears about having to endure constant teasing or "male " ambiance.<br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<h3> From a survey</h3><br />
<p> &nbsp;&nbsp;<br />
Finally, a survey was conducted among iGEMers and former iGEMers to understand their motivations and activities in iGEM. The study was designed to be unbiased and to avoid stereotype threat (for example by putting the question about gender in the end among many other pieces of information). It is still available <a href="http://bit.ly/14WykuZ"> here</a>. Participants in the survey had to rank from 1 to 5 (1 being not important, and 5 very important) answers to questions regarding personnal and professional motivations for participating in iGEM as well values and on what did they spend their time. 63 people answered among whom 32% were women.</p><br />
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<center><br />
<img src="https://static.igem.org/mediawiki/2013/c/c5/PB_GS_Survey1.png" width="500px" /><br />
<b>Figure 9. </b> Results of survey : what did you hope to learn in iGEM ?<br />
</center><br />
<br><br><br />
<p><br />
It is interesting to notice that men and women answered almost exactly the same way regarding most of the questions. Women gave a little more importance for the value of fundamental research in iGEM while men graded a bit better "Changing the world". Motivations were approximately the same as well as time spent on each activity. Just a little fact was that men considered human practices a bit more important than women did but spent a little less time on it. <br />
There is only one main difference (more than one point out of five which is represented below) : the will to lead a project and lead a team. It is striking to see how much men are more motivated to lead teams than women. This is definitely to put in relation with the number of women advisers found and the impact it can then have on teams mixity. This could reflect women lack of self esteem in some parts of their work.</p><br />
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<div id="Recommendations"></div><br />
<h2> Recommendations</h2><br />
<div class="leftparagraph"><br />
<p> &nbsp;&nbsp;<br />
Considering all the results that were presented above, here is a list of recommendations for the iGEM foundation to pursue an active policy to improve mixity in iGEM.<br />
<ul><br />
<li> Raise the number of women judges </li> <br />
<li> Promote large teams </li> <br />
<li> Write up a small paragraph to team heads to insist on the importance of motivating young women to be advisers.</li><br />
<li> Giving Bonus point when the team have women advisers </li><br />
</ul><br />
</p><br />
</div><br />
<div class="rightparagraph"><br />
<p>And finally, add in iGEM requirements a Gender reflection. By having teams filling out the database that was built and answering the survey and write a small paragraph about how they see mixity in their team and what it could bring, it would drastically raise the awareness of young men and women about the gender problem in science. Having an up-to-date database is also a great way to see improvements in a quantitative manner. It would allow a direct assessment of the effects of an active gender policy which would be a unique example in science. iGEM could become a leader in that fight and prepare the new generation of scientists to finally get rid of the gender inequality in science<br />
</p><br />
<br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2>Litterature</h2><br />
<div class="leftparagraph"><br />
<ul><br />
<li>P. Allotey, M. Gyapong Gender in tuberculosis research INT J TUBERC LUNG DIS 2008 </li><br />
<li>M. Calid, S. Rasul, S Ullah Khan, M; Saeed Gender differences in delay to s to tuberculosis diagnosis and treatment outcome<br><br />
European Commission She figures 2012<br><br />
European Commission, Mapping the gaze : getting more women to the top in Research 2008.<br><br />
</li><br />
<li>C.B. Holmes, H. Hausler, P. Hunn : A review of sex differences in the epidemiology of tuberculosis</li><br />
<li>A. N. Martinez J. T. Rhee, P. M. Small,‡M. A. Behr Sex differences in the epidemiology of tuberculosis in San Francisco INT J TUBERC LUNG DIS 4(1):26–31 2000</li><br />
<li>Moss-Racusin et al, (2012) Science faculty’s subtle gender biases favor male students PNAS </li><br />
<br />
</ul><br />
</div><br />
<div class="rightparagraph"><br />
<ul><br />
<li>McKinsey and Company Women Matter, 2007 </li><br />
<li>Olivier Neyrolles, Lluis Quintana-Murci Sexual Inequality in Tuberculosis, Plos Medicine 2009</li><br />
<li>Nosek et al. (2009) National differences in gender–science stereotypes predict national sex differences in science and math achievement. PNAS June 30, 2009 vol. 106 no. 26 10593–10597</li><br />
<br />
<li>E. Pollack Why Are There Still So Few Women in Science? NY TImes OCtober 2013</li><br />
<li>Al S. Rhines The role of sex differences in the prevalence and transmission of tuberculosis : Tuberculosis 2013</li><br />
<li>M. W. Uplekar, S. Rangan, M. G. Weiss, J. Ogden, M. W. Borgdorff, P. Hudelson Attention to gender issues in tuberculosis control INT J TUBERC LUNG DIS 4(1):26–31 2001</li><br />
</ul><br />
</div><br />
<div style="clear: both;"></div><br />
<h2>Attributions</h2><br />
<p>We would like to thank Flora Vincent, President of <a href="http://wax-science.fr/")>WAX Science</a> association for her precious help in analyzing the results, and Kim de Mora and Kitwa from the iGEM foundation, for helping spreading the survey. <p><br />
<p>This project was designed and accomplished by Aude Bernheim, Clovis Basier, Matt Deyell, Marguerite Benony and Sebastian Jaramillo in consultation with Edwin Wintermute and Ariel Lindner.</p><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Yzegmanhttp://2013.igem.org/File:GS_Role.pngFile:GS Role.png2013-10-28T21:46:08Z<p>Yzegman: </p>
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<div></div>Yzegmanhttp://2013.igem.org/File:GS_Region.pngFile:GS Region.png2013-10-28T21:45:18Z<p>Yzegman: </p>
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<div></div>Yzegmanhttp://2013.igem.org/File:GS_Prize.pngFile:GS Prize.png2013-10-28T21:45:01Z<p>Yzegman: uploaded a new version of &quot;File:GS Prize.png&quot;</p>
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<div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T21:38:47Z<p>Yzegman: </p>
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<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
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<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
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<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
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<div class="biocriks"><br />
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<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
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<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
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<div class="biocriks"><br />
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<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
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<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
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<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
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<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
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<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
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<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
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<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
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<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
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<h2><a href="">Modelling</a></h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
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<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
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<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
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<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
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<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
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<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<br><center><img src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br><br />
<b>Gender balance and success in iGEM</b><br>Winning teams have a significantly higher number of women and are more Gender balanced.<br />
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<div class="subbox3" style="margin-right:0"><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
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<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T21:33:58Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
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<div id="grouptitle"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<style><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
<style><br />
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<div class="subbox3"><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<br><center><img src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"></center><br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T21:32:41Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
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}<br />
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.subbox1 {<br />
width:265px;<br />
margin-right:15px;<br />
}<br />
.bkgr, .aims {<br />
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<div id="grouptitle"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
<style><br />
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<div class="overbox"><br />
<div class="subbox3"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.<br />
<img src="https://static.igem.org/mediawiki/2013/d/de/GS_Prize.png"><br />
</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/File:GS_Prize.pngFile:GS Prize.png2013-10-28T21:30:35Z<p>Yzegman: uploaded a new version of &quot;File:GS Prize.png&quot;</p>
<hr />
<div></div>Yzegmanhttp://2013.igem.org/File:GS_Prize.pngFile:GS Prize.png2013-10-28T21:30:07Z<p>Yzegman: </p>
<hr />
<div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T20:30:43Z<p>Yzegman: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
<html><br />
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<div id="grouptitle"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
</div><br />
<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
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<br />
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</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img height="50%" src="https://static.igem.org/mediawiki/2013/b/b2/Newfiguresabotagepresentation.png"/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
</p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
</div><br />
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<br />
<br />
</div></div>Yzegmanhttp://2013.igem.org/Team:Paris_Bettencourt/ResultsTeam:Paris Bettencourt/Results2013-10-28T20:27:21Z<p>Yzegman: </p>
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<div>{{:Team:Paris_Bettencourt/Wiki}}<br />
{{:Team:Paris_Bettencourt/Menu}}<br />
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<img src="https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png" width="1100"/><br />
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<div id="page"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li><br />
<li> Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette. </li><br />
<li>Testing the new assembly standard for our cloning.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/a/ab/Bb_detect_cas9.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/9/97/Bb_detect_crRNAKan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/7b/Bb_detect_gRNAkan.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank"><img width="24%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/05/Bb_detect_LacZ.png"/></a><br />
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<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="80%" src="https://static.igem.org/mediawiki/2013/0/01/PB_13_Results_detect_killing_assay.png"/></center><br />
<br><br />
<br />
<p style="margin-top:-60px;font-size:13px"><b>CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. </b>We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>SirA is an essential gene in latent tuberculosis infections. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To perform a drug screen targeted at the sirA gene from mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media.</li><br />
<li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis.</li><br />
<li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural analysis.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137000" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/7/79/Bb_target_sirA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137001" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/c/cb/Bb_target_fprA.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137002" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/4/40/Bb_target_fdxA.png"/></a><br />
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<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center><br />
<p style="font-size:13px"> <b>MycoSIR E. coli depend on our synthetic pathway for growth.</b> E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth <br />
</p><br />
</div><br />
</div><br />
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<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics. </p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li><br />
<li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li><br />
<li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li><br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank"><img height="80%" style="margin-top:15px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/a/a0/Bb_infiltrate_tdmh.png"/></a><br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center><br />
<p style="font-size:13px"> <b>TDMH expression kills mycobacteria in culture.</b> We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.<br />
</p><br />
</div><br />
</div><br />
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<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2><br />
<div class="overbox"><br />
<div class="subbox1"><br />
<div class="bkgr"><br />
<h2>Background</h2><br />
<p>One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.</p><br />
</div><br />
<div class="aims"><br />
<h2>Aims</h2><br />
<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.</p><br />
</div><br />
</div><br />
<div class="subbox2"><br />
<div class="results"><br />
<h2>Results</h2><br />
<ul><br />
<li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.</li><br />
<li>Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.</li><br />
<li>Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).</li><br />
<br />
</ul><br />
</div><br />
<div class="biocriks"><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank"><img height="80%" style="margin-top:15px;margin-left:30px" src="https://static.igem.org/mediawiki/2013/3/37/Bb_sabotage_aKAN.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/6/66/Bb_sabotage_aCm.png"/></a><br />
<br />
<a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank"><img height="80%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/7/72/Bb_sabotage_aLac.png"/></a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</div><br />
<div class="mainfig"><br />
<center><br> <img width="95%" src="https://2013.igem.org/File:Newfiguresabotagepresentation.png "/></center><br />
<p style="font-size:13px"><b>Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. </b>The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.<br />
</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
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<h2><a href="">Modelling</a></h2><br />
<div class="overbox"><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2><br />
<p>This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.</p><br />
</div><br />
<div class="projtile"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural Analysis of SirA</a></h2><br />
<p>Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure. </p><br />
</div><br />
<div class="projtile" style="margin-right:0;"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2><br />
<p>We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.</p><br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology Transfer <img height="100%" src="https://static.igem.org/mediawiki/2013/6/6d/PB_logotransferttechno.png"/></a></h2><br />
<p> An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France <img height="100%" src="https://static.igem.org/mediawiki/2013/c/cf/PB_logofrance.png"/></a></h2><br />
<p>Analysis of the social, medical and political aspects of the management of tuberculosis in France.<br />
Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics. </p><br />
</div><br />
</div><br />
<div class="subbox4"><br />
<div class="aims" style="height:100%"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study <img height="100%" src="https://static.igem.org/mediawiki/2013/d/da/PB_logogender.png"/></a></h2><br />
<p>A comprehensive and quantitative study of gender equality(equality) in iGEM and synthetic biology. A database was gathered to depict sex ratio in teams' students and supervisors in all iGEM teams as well as other available information. This was statistically analysed to investigate gender in(equality) in iGEM, as well as SB conferences and synthetic biology labs.</p><br />
</div> <br />
</div><br />
<br />
<div class="subbox3" style="margin-right:0"><br />
<div class="bkgr"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB Facts <img height="100%" src="https://static.igem.org/mediawiki/2013/6/69/PB_logoTBfacts.png"/></a></h2><br />
<p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p><br />
</div><br />
<div class="aims"><br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB Gallery <img height="100%" src="https://static.igem.org/mediawiki/2013/d/de/PB_logoTBgallery.png"/><a/></h2><br />
<p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p><br />
</div> <br />
</div><br />
</div><br />
<div style="clear: both;"></div><br />
<br />
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2><br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">SensiGEM</a></h2><br />
<p>SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.<br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2><br />
<p> A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot. <br />
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<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2><br />
<p>Idea, bibliography, and beer sharing!</p> <br />
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</div></div>Yzegmanhttp://2013.igem.org/File:Newfiguresabotagepresentation.pngFile:Newfiguresabotagepresentation.png2013-10-28T20:25:49Z<p>Yzegman: </p>
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<div></div>Yzegmanhttp://2013.igem.org/File:Bb_detect_LacZ.pngFile:Bb detect LacZ.png2013-10-28T20:02:35Z<p>Yzegman: uploaded a new version of &quot;File:Bb detect LacZ.png&quot;</p>
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<div></div>Yzegmanhttp://2013.igem.org/File:Bb_target_sirA.pngFile:Bb target sirA.png2013-10-28T19:58:30Z<p>Yzegman: uploaded a new version of &quot;File:Bb target sirA.png&quot;</p>
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<div></div>Yzegman