Team:Paris Bettencourt/YonatanTest

From 2013.igem.org

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    <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2>
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  <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2>
     <div class="overbox">
     <div class="overbox">
       <div class="subbox1">
       <div class="subbox1">
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</div>
<div class="biocriks">
<div class="biocriks">
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  <ol>
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  <center> <img width="95%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/5/55/PB_Bb_target.png"/><br></center>
-
            <li><a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank">BBa_K1137012 (gRNA anti-KAN)</a></li>
+
-
            <li><a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank">BBa_K1137013 (crRNA anti-KAN)</a></li>
+
-
            <li><a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank">BBa_K1137014 (tracrRNA-Cas9)</a></li>
+
-
            <li><a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank">BBa_K1137015 (pRecA-LacZ)</a></li>
+
-
  </ol>
+
</div>
</div>
       </div>
       </div>
       <div class="mainfig">
       <div class="mainfig">
-
         <center> <img width="90%" src="https://static.igem.org/mediawiki/2013/1/1f/PB_phagesensor10.png"/><br></center>
+
         <center> <img height="60%" src="https://static.igem.org/mediawiki/2013/9/99/PB_fig_target.png"/></center>
-
         <p>After expression of the Cas9 and gRNA, the gRNA guides the Cas9 to the target sequence, the kanamycin resistance. There, the Cas9 generates a double strand break. This activates the SOS response. The reporter LacZ is under the pRECA promoter, which gets activated during the SOS response and we get hence a blue cell, if the resistance gene has successfully been detected.</p>
+
        <p style="margin-top:-60px;font-size:13px">CRISPR anti-Kan plasmids target kanamycin resistant E. coli. WT (blue) and a kanamycin resistant strain (KanR, red) were co-transformed with a plasmid carrying the Cas9 construct, and a second plasmid carrying the anti-Kanamycin gRNA. WT was successfully transformed with one or both plasmids.  KanR E. coli couldn’t be tranformed with both plasmids because of Cas9-induced cleavage of the chromosome specifically at the KanR cassette, with about 99% efficiency.
 +
</p>
 +
      </div>
 +
    </div>
 +
    <div style="clear: both;"></div>
 +
   
 +
    <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2>
 +
    <div class="overbox">
 +
      <div class="subbox1">
 +
<div class="bkgr">
 +
  <h2>Background</h2>
 +
  <p>SirA is an essential gene in latent tuberculosis infections  </p>
 +
</div>
 +
<div class="aims">
 +
  <h2>Aims</h2>
 +
  <p>To perform an drug screen targeted at the sirA gene from mycobacteria</p>
 +
</div>
 +
      </div>
 +
      <div class="subbox2">
 +
<div class="results">
 +
  <h2>Results</h2>
 +
  <ul>
 +
            <li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li>
 +
            <li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li>
 +
            <li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li>
 +
  </ul>
 +
</div>
 +
<div class="biocriks">
 +
  <center> <img width="95%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/0e/PB_bb_target1.png"/><br></center>
 +
</div>
 +
      </div>
 +
      <div class="mainfig">
 +
        <center> <img width="100%" src="https://static.igem.org/mediawiki/2013/3/31/PB_fig_target1.png"/></center>
 +
         <p style="font-size:13px"> MycoSIR E. coli depend on our synthetic pathway for growth. 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
 +
</p>
 +
      </div>
 +
    </div>
 +
    <div style="clear: both;"></div>
 +
   
 +
    <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2>
 +
    <div class="overbox">
 +
      <div class="subbox1">
 +
<div class="bkgr">
 +
  <h2>Background</h2>
 +
  <p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics.  </p>
 +
</div>
 +
<div class="aims">
 +
  <h2>Aims</h2>
 +
  <p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p>
 +
</div>
 +
      </div>
 +
      <div class="subbox2">
 +
<div class="results">
 +
  <h2>Results</h2>
 +
  <ul>
 +
            <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>
 +
            <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>
 +
            <li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li>
 +
  </ul>
 +
</div>
 +
<div class="biocriks">
 +
  <center> <img width="25%" style="margin-top:15px" src="https://static.igem.org/mediawiki/2013/0/00/PB_bb_Infil.png"/><br></center>
 +
</div>
 +
      </div>
 +
      <div class="mainfig">
 +
        <center><br> <img width="95%" src="https://static.igem.org/mediawiki/2013/b/bb/PB_fig_Infil.png"/></center>
 +
        <p style="font-size:13px"> FTDMH expression kills mycobacteria in culture. 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.
 +
</p>
 +
      </div>
 +
    </div>
 +
    <div style="clear: both;"></div>
 +
 
 +
    <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2>
 +
    <div class="overbox">
 +
      <div class="subbox1">
 +
<div class="bkgr">
 +
  <h2>Background</h2>
 +
  <p>One of the main concern about tuberculosis today is the emergence of antibiotic resistant strain</p>
 +
</div>
 +
<div class="aims">
 +
  <h2>Aims</h2>
 +
  <p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to those same antibiotics.</p>
 +
</div>
 +
      </div>
 +
      <div class="subbox2">
 +
<div class="results">
 +
  <h2>Results</h2>
 +
  <ul>
 +
            <li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins</li>
 +
            <li>successful conversion of antibiotic resistant population of E. coli to a sensitive state</li>
 +
  </ul>
 +
</div>
 +
<div class="biocriks">
 +
  <center> <img width="95%" style="margin-top:10px" src="https://static.igem.org/mediawiki/2013/e/e5/PB_bb_Sabotage.png"/><br></center>
 +
</div>
 +
      </div>
 +
      <div class="mainfig">
 +
        <center><br> <img height="40%" src="https://static.igem.org/mediawiki/2013/3/3c/PB_fig_Sabotage.png  "/></center>
 +
        <p style="font-size:13px">Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. WT and chloramphenicol resitant strains (1 mL at OD 0.7) were infected with 10 ul of collected phage. Cells were plated at various antibiotic concentrations to measure sensitivity. (A) The anti-Cm phage system effectively killed 99.1% of the population at 1 mg/mL of Chloramphenicol. (B) Of the surviving cells, 70% still carried the GFP phage marker. This suggests that 70% of system failure originates from resistance to the siRNA, and 30 originates from resistance to the phage.
 +
</p>
 +
      </div>
 +
    </div>
 +
    <div style="clear: both;"></div>
 +
 
 +
    <style>
 +
      .projtile {
 +
        height:480px;
 +
        margin-right:15px;
 +
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 +
    <h2><a href="">Modelling</a></h2>
 +
    <div class="overbox">
 +
      <div class="projtile">
 +
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Population Dynamics Model</a></h2>
 +
<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>
 +
      </div>
 +
      <div class="projtile">
 +
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Structural analysis of SirA</a></h2>
 +
<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>
 +
      </div>
 +
      <div class="projtile" style="margin-right:0;">
 +
<h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Flux Balance Analysis</a></h2>
 +
<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>
 +
      </div>
 +
    </div>
 +
    <div style="clear: both;"></div>
 +
 
 +
    <h2><a href="https://2013.igem.org/wiki/index.php?title=Team:Paris_Bettencourt/Human_Practice/Overview">Human Practice</a></h2>
 +
    <style>
 +
      .subbox3 {
 +
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 +
      height:480px;   
 +
      width:320px;   
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 +
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 +
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 +
      float:left;
 +
      height:480px;   
 +
      width:425px;
 +
      margin-right:15px;
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 +
    </style>
 +
    <div class="overbox">
 +
      <div class="subbox3">
 +
<div class="bkgr">
 +
  <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Technology_Transfer">Technology  Transfer</a></h2>
 +
  <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>
 +
        </div>
 +
<div class="aims">
 +
  <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_France">TB in France</a></h2>
 +
  <p>An analysis of the social and political aspects of the management of Tuberculosis in France.</p>
 +
        </div>
 +
      </div>
 +
      <div class="subbox4">
 +
<div class="aims" style="height:100%">
 +
  <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/Gender_Study">Gender Study</a></h2>
 +
          <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>
 +
        </div>       
 +
      </div>
 +
      <div class="subbox3" style="margin-right:0">
 +
<div class="bkgr">
 +
  <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Facts">TB facts</a></h2>
 +
  <p> Infographics page containing TB data and facts that captures all you need to know at a glance.</p>
 +
        </div>
 +
<div class="aims">
 +
  <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Human_Practice/TB_Gallery"> TB gallery<a/></h2>
 +
  <p> A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .</p>
 +
        </div>
 +
      </div>
 +
    </div>
 +
    <div style="clear: both;"></div>
 +
 
 +
    <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Collaboration</a></h2>
 +
    <div class="overbox">
 +
      <div class="projtile">
 +
        <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/SensiGEM">Sensigem</a></h2>
 +
        <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.
 +
        </p>
 +
      </div>
 +
      <div class="projtile">
 +
        <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">BGU iGEM Team from Israel</a></h2>
 +
        <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. 
 +
        </p>
 +
      </div>
 +
      <div class="projtile" style="margin-right:0;">
 +
        <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Collaboration">Braunschweig iGEM Team</h2>
 +
        <p>Idea, bibliography, and beer sharing!</p>       
       </div>
       </div>
     </div>
     </div>
     <div style="clear: both;"></div>
     <div style="clear: both;"></div>
-
       
+
 
 +
 
   </div>
   </div>
    
    

Latest revision as of 15:38, 26 October 2013

Detect

Background

CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing.

Aims

Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.

Results

  • Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks.
  • Testing the new assembly standard for our cloning.

CRISPR anti-Kan plasmids target kanamycin resistant E. coli. WT (blue) and a kanamycin resistant strain (KanR, red) were co-transformed with a plasmid carrying the Cas9 construct, and a second plasmid carrying the anti-Kanamycin gRNA. WT was successfully transformed with one or both plasmids. KanR E. coli couldn’t be tranformed with both plasmids because of Cas9-induced cleavage of the chromosome specifically at the KanR cassette, with about 99% efficiency.

Target

Background

SirA is an essential gene in latent tuberculosis infections

Aims

To perform an drug screen targeted at the sirA gene from mycobacteria

Results

  • Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media
  • Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis
  • Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis

MycoSIR E. coli depend on our synthetic pathway for growth. 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

Infiltrate

Background

Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics.

Aims

To create an E. coli strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.

Results

  • We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in E.coli and showed that it is highly toxic to mycobacteria in culture.
  • We expressed the lysteriolyin O (LLO) gene in E. coli and showed that it is capable of entering the macrophage cytosol.
  • We co-infected macrophages with both mycobacteria and our engineered E. coli to characterize the resulting phagocytosis and killing.


FTDMH expression kills mycobacteria in culture. 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.

Sabotage

Background

One of the main concern about tuberculosis today is the emergence of antibiotic resistant strain

Aims

Our objective is to make an antibiotic-resistant bacterial population sensitive again to those same antibiotics.

Results

  • Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins
  • successful conversion of antibiotic resistant population of E. coli to a sensitive state


Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. WT and chloramphenicol resitant strains (1 mL at OD 0.7) were infected with 10 ul of collected phage. Cells were plated at various antibiotic concentrations to measure sensitivity. (A) The anti-Cm phage system effectively killed 99.1% of the population at 1 mg/mL of Chloramphenicol. (B) Of the surviving cells, 70% still carried the GFP phage marker. This suggests that 70% of system failure originates from resistance to the siRNA, and 30 originates from resistance to the phage.

Modelling

Population Dynamics Model

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

Structural analysis of SirA

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.

Flux Balance Analysis

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.

Human Practice

Technology Transfer

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.

TB in France

An analysis of the social and political aspects of the management of Tuberculosis in France.

Gender Study

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.

TB facts

Infographics page containing TB data and facts that captures all you need to know at a glance.

TB gallery

A gallery of famous historic figures who had tuberculosis, made to raise awareness to its prevalence of in the past and present .

Collaboration

Sensigem

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.

BGU iGEM Team from Israel

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.

Braunschweig iGEM Team

Idea, bibliography, and beer sharing!

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
+33 1 44 41 25 22/25
team2013@igem-paris.org
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