Difference between revisions of "Team:ETH Zurich/Basic Part"

 
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<h1 class="headline">Basic Parts</h1>
 
<h1 class="headline">Basic Parts</h1>
  
−
<figure class="biobrick">
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<figure class="top-fig-single">
−
     <img src="https://static.igem.org/mediawiki/2017/7/7e/T--ETH_Zurich--basicparts.png" alt="FIXME">
+
     <img src="https://static.igem.org/mediawiki/2017/b/b5/T--ETH_Zurich--basicpartspage.png" alt="FIXME">
 
</figure>
 
</figure>
  
−
<section class="overview">
+
<section class="query">
−
<h1>Overview</h1>
+
    <p><em>You can read details about the design of our Basic Parts here. To check out all the parts that we created, visit <a href="https://2017.igem.org/Team:ETH_Zurich/Parts">Parts.</a></em></p>
−
  <p>All parts were codon-optimized for expression in <i>E. coli</i> Nissle with Geneious and modified to remove forbidden restriction sites.</p>
+
</section>
  
−
 
+
<section>
−
  <table width="100%">
+
    <h1>Overview</h1>
 +
    <table width="100%">
 
         <tr>
 
         <tr>
 
             <!--<th>Design</th>-->
 
             <!--<th>Design</th>-->
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             <th>BioBrick</th>
 
             <th>BioBrick</th>
 
         </tr>
 
         </tr>
−
      <tr>
+
        <tr>
 
             <!--<td>Image</td>-->
 
             <!--<td>Image</td>-->
 
             <td><strong>Best Basic Part:</strong><br>AND Gate B</td>
 
             <td><strong>Best Basic Part:</strong><br>AND Gate B</td>
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         <tr>
 
         <tr>
 
             <td>Azurin</td>
 
             <td>Azurin</td>
−
             <td>Redox protein originating from <span class="bacterium">P. aeruginosa</span> with anti-cancer activity</td>
+
             <td>Redox protein originating from <span class="bacterium">P. aeruginosa</span> with cytotoxic activity</td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500001">BBa_K2500001</a></td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500001">BBa_K2500001</a></td>
 
         </tr>
 
         </tr>
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         <tr>
 
         <tr>
 
             <!--<td>Image</td>-->
 
             <!--<td>Image</td>-->
−
             <td>p<sub>TlpA</sub></td>
+
             <td>P<sub>TlpA</sub></td>
 
             <td>Temperature-responsive promoter optimized for slight activation above 37°C and full activation at 45 °C</td>
 
             <td>Temperature-responsive promoter optimized for slight activation above 37°C and full activation at 45 °C</td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500003">BBa_K2500003</a></td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500003">BBa_K2500003</a></td>
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             <td><a href="http://parts.igem.org/Part:BBa_K2500004">BBa_K2500004</a></td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500004">BBa_K2500004</a></td>
 
         </tr>
 
         </tr>
−
      <tr>
+
        <tr>
 
             <!--<td>Image</td>-->
 
             <!--<td>Image</td>-->
 
             <td>RBS_TlpA</td>
 
             <td>RBS_TlpA</td>
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             <td><a href="http://parts.igem.org/Part:BBa_K2500005">BBa_K2500005</a></td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500005">BBa_K2500005</a></td>
 
         </tr>
 
         </tr>
−
      <tr>
+
        <tr>
 
             <!--<td>Image</td>-->
 
             <!--<td>Image</td>-->
 
             <td>Protein E</td>
 
             <td>Protein E</td>
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             <td><a href="http://parts.igem.org/Part:BBa_K2500006">BBa_K2500006</a></td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500006">BBa_K2500006</a></td>
 
         </tr>
 
         </tr>
−
<tr>
+
        <tr>
 
             <!--<td>Image</td>-->
 
             <!--<td>Image</td>-->
 
             <td>RBS<sub>eng</sub>_TlpA</td>
 
             <td>RBS<sub>eng</sub>_TlpA</td>
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             <td><a href="http://parts.igem.org/Part:BBa_K2500010">BBa_K2500010</a></td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500010">BBa_K2500010</a></td>
 
         </tr>
 
         </tr>
−
+
 
 
         <tr>
 
         <tr>
−
            <!--<td>Image</td>-->
 
 
             <td>AND Gate C</td>
 
             <td>AND Gate C</td>
 
             <td>Synthetic promoter responsive to LldR and luxR </td>
 
             <td>Synthetic promoter responsive to LldR and luxR </td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500012">BBa_K2500012</a></td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500012">BBa_K2500012</a></td>
 
         </tr>
 
         </tr>
−
      <tr>
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        <tr>
−
             <td>p<sub>Const</sub>_RBS_LldP/<br>LldR_p<sub>Const</sub>_RBS_LuxR</td>
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             <td>P<sub>Const</sub>_RBS_LldP/<br>LldR_P<sub>Const</sub>_RBS_LuxR</td>
 
             <td>Expression cassette consisting of LuxR and LldP/LldR</td>
 
             <td>Expression cassette consisting of LuxR and LldP/LldR</td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500013">BBa_K2500013</a></td>
 
             <td><a href="http://parts.igem.org/Part:BBa_K2500013">BBa_K2500013</a></td>
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</section>
 
</section>
  
−
<section class="design">
+
<section>
−
<h1>Design</h1>
+
    <h1>Design</h1>
 +
    <p>All parts were codon-optimized for expression in <span class="bacterium">E. coli</span> Nissle with Geneious and modified to remove forbidden restriction sites.</p>
 +
    <div class="multi-summary">
 +
        <details>
 +
            <summary>BBa_K2500000: Bacterioferritin</summary>
 +
            <p><strong>Design notes:</strong> Bacterioferritins are bacterial iron storage proteins. It has been shown that overexpression of bacterioferritin in <i>E. coli</i> Nissle 1917 can lead to a visible contrast change in MRI, which allows for visualization of the bacteria. We used a heme-deletion mutant as it does not decrease contrast change.</p>
 +
            <figure class="bfrmodel">
 +
                <img src="https://static.igem.org/mediawiki/2017/6/6e/T--ETH_Zurich--bfrmodel.png">
 +
            </figure>
 +
            <p><strong>Organism:</strong> <span class="bacterium">Escherichia coli</span> Nissle</p>
 +
            <p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K1438001">BBa_K1438001</a></p>
 +
            <p><strong>Source:</strong> gBlock</p>
 +
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500000">BBa_K2500000</a></p>
 +
        </details>
  
−
  <div class="multi-summary">
+
        <details>
 +
            <summary>BBa_K2500001: Azurin</summary>
 +
            <p><strong>Design notes:</strong> Azurin is a redox protein with the ability to induce apoptosis in mammalian cells upon internalization. We removed the native signal peptide which is part of the pre-protein form of azurin as no targeting within the bacterial host cells is required.</p>
 +
            <figure class="azurinmodel">
 +
                <img src="https://static.igem.org/mediawiki/2017/4/4d/T--ETH_Zurich--azurin.png">
 +
            </figure>
 +
            <p><strong>Organism:</strong> <span class="bacterium">Pseudomonas aeruginosa</span></p>
 +
            <p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K835004">BBa_K835004</a></p>
 +
            <p><strong>Source:</strong> gBlock</p>
 +
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500001">BBa_K2500001</a></p>
 +
        </details>
  
−
<details>
+
        <details>
−
<summary>BBa_K2500000: Bacterioferritin</summary>
+
            <summary>BBa_K2500002: p28</summary>
−
<p><strong>Design notes:</strong> Bacterioferritins are bacterial iron storage proteins. It has been shown that overexpression of bacterioferritin in <i>E. coli</i> Nissle 1917 can lead to a visible contrast change in MRI, which allows for visualization of the bacteria. We used a heme-deletion mutant as the absence of heme does not influence the contrast change and results in stronger iron retention within the protein scaffold.</p>
+
            <p><strong>Design notes:</strong> The effector domain of azurin comprises amino acids 50 to 77. We extracted the sequence, introduced a start codon (ATG) at the beginning and two stop codons (TAA TAA) at the end. By encoding only the effector, we were aiming at achieving faster expression levels and a higher cytotoxic load carried by the bacteria.</p>
−
<figure class="bfrmodel">
+
            <figure class="p28model">
−
  <img src="https://static.igem.org/mediawiki/2017/6/6e/T--ETH_Zurich--bfrmodel.png">
+
                <img src="https://static.igem.org/mediawiki/2017/e/e2/T--ETH_Zurich--p28model.png">
−
</figure>
+
            </figure>
−
<p><strong>Organism:</strong> <span class="bacterium">Escherichia coli</span> Nissle</p>
+
            <p><strong>Organism:</strong> <span class="bacterium">Pseudomonas aeruginosa</span></p>
−
<p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K1438001">BBa_K1438001</a></p>
+
            <p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K2500001">BBa_K2500001</a></p>
−
<p><strong>Source:</strong> gBlock</p>
+
            <p><strong>Source:</strong> gBlock</p>
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500000">BBa_K2500000</a></p>
+
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500002">BBa_K2500002</a></p>
−
</details>
+
        </details>
  
−
<details>
+
        <details>
−
<summary>BBa_K2500001: Azurin</summary>
+
            <summary>BBa_K2500003: p<sub>TlpA</sub></summary>
−
<p><strong>Design notes:</strong> Azurin is a redox protein with the ability to induce apoptosis in human cells upon internalization. We removed the native signal peptide which is part of the pre-protein form of azurin as no targeting within the bacterial host cells is required.</p>
+
            <p><strong>Design notes:</strong> The p<sub>TlpA</sub> promoter regulates the lysis step of CATE. The promoter is bound and inhibited by TlpA repressor proteins below 37 °C, thereby blocking the release of the cytotoxic compound at body temperature. To fully activate the promoter and thus cell lysis, the desired area is heated to 45 °C with focused ultrasound.</p>
−
<figure class="azurinmodel">
+
            <figure class="TlpAaction">
−
  <img src="https://static.igem.org/mediawiki/2017/4/4d/T--ETH_Zurich--azurin.png">
+
                <img src="https://static.igem.org/mediawiki/2017/e/eb/T--ETH_Zurich--tlpadescription.png">
−
</figure>
+
            </figure>
−
<p><strong>Organism:</strong> <span class="bacterium">Pseudomonas aeruginosa</span></p>
+
            <p><strong>Organism:</strong> <span class="bacterium">Salmonella typhimurium</span></p>
−
<p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K835004">BBa_K835004</a></p>
+
            <p><strong>Based on:</strong> <a href="https://doi.org/10.1038/nchembio.2233">Piraner, Dan I., et al. <cite>Nature Chemical Biology</cite> 13.1 (2017): 75-80.</a></p>
−
<p><strong>Source:</strong> gBlock</p>
+
            <p><strong>Source:</strong> oligonucleotide sequence</p>
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500001">BBa_K2500001</a></p>
+
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500003">BBa_K2500003</a></p>
−
</details>
+
        </details>
  
−
<details>
+
        <details>
−
<summary>BBa_K2500002: p28</summary>
+
            <summary>BBa_K2500004: TlpA</summary>
−
<p><strong>Design notes:</strong> Designed by taking amino acids 50 to 77 from azurin and adding a start codon (ATG) at the beginning and two stop codons (TAA TAA) at the end.</p>
+
            <p><strong>Design notes:</strong> TlpA is a constitutively expressed repressor protein which inhibits transcription of genes downstream of the TlpA promoter by binding to it. The binding to the promoter is reversibly deactivated by temperatures above 37 °C with full deactivation at 45 °C.</p>
−
<p><strong>Organism:</strong> <span class="bacterium">Pseudomonas aeruginosa</span></p>
+
            <figure class="TlpAaction">
−
<p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K2500001">BBa_K2500001</a></p>
+
                <img src="https://static.igem.org/mediawiki/2017/e/eb/T--ETH_Zurich--tlpadescription.png">
−
<p><strong>Source:</strong> gBlock</p>
+
            </figure>
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500002">BBa_K2500002</a></p>
+
            <p><strong>Organism:</strong> <span class="bacterium">Salmonella typhimurium</span></p>
−
</details>
+
            <p><strong>Based on:</strong> <a href="https://doi.org/10.1038/nchembio.2233">Piraner, Dan I., et al. <cite>Nature Chemical Biology</cite> 13.1 (2017): 75-80.</a></p>
 +
            <p><strong>Source:</strong> gBlock</p>
 +
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500004">BBa_K2500004</a></p>
 +
        </details>
  
−
<details>
+
        <details>
−
<summary>BBa_K2500003: p<sub>TlpA</sub></summary>
+
            <summary>BBa_K2500005: RBS_TlpA</summary>
−
<p><strong>Organism:</strong> <span class="bacterium">Salmonella typhimurium</span></p>
+
            <p><strong>Design notes:</strong> Parameters influencing the final expression levels of the genes downstream of pTlpA include the RBS of its repressor protein TlpA. By tweaking these sequences, it should be possible to obtain a wide range of leakiness and fold changes (fold-change up to 200-fold).</p>
−
<p><strong>Based on:</strong> <a href="https://doi.org/10.1038/nchembio.2233">Piraner, Dan I., et al. <cite>Nature Chemical Biology</cite> 13.1 (2017): 75-80.</a></p>
+
            <figure class="RBS_TlpA">
−
<p><strong>Source:</strong> oligonucleotide sequence</p>
+
                <img src="https://static.igem.org/mediawiki/2017/5/57/T--ETH_Zurich--rbs_tlpA.png">
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500003">BBa_K2500003</a></p>
+
            </figure>
−
</details>
+
            <p><strong>Organism:</strong> <span class="bacterium">Salmonella typhimurium</span></p>
 +
            <p><strong>Based on:</strong> <a href="https://doi.org/10.1038/nchembio.2233">Piraner, Dan I., et al. <cite>Nature Chemical Biology</cite> 13.1 (2017): 75-80.</a></p>
 +
            <p><strong>Source:</strong> gBlock</p>
 +
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500005">BBa_K2500005</a></p>
 +
        </details>
  
−
<details>
+
        <details>
−
<summary>BBa_K2500004: TlpA</summary>
+
            <summary>BBa_K2500006: Protein E</summary>
−
<p><strong>Design notes: </strong>TlpA is a constitutively expressed repressor protein which inhibits transcription of genes downstream of the TlpA promoter by binding to it. The binding to the promoter is reversibly deactivated by temperatures above 37 °C with full deactivation at 45 °C.</p>
+
            <p><strong>Design notes:</strong> Protein E is a lysis protein originally encoded by the bacteriophage Phi X 147. It is now generally accepted that the most probable cellular target of protein E is the enzyme translocase I, encoded by the mraY gene. Translocase I is required for cell wall biosynthesis and its inhibition results to cell lysis.</p>
−
<figure class="TlpAaction">
+
            <figure class="proteinE">
−
  <img src="https://static.igem.org/mediawiki/2017/e/eb/T--ETH_Zurich--tlpadescription.png">
+
                <img src="https://static.igem.org/mediawiki/2017/3/36/T--ETH_Zurich--proteinE.png">
−
</figure>
+
            </figure>
−
<p><strong>Organism:</strong> <span class="bacterium">Salmonella typhimurium</span></p>
+
            <p><strong>Organism:</strong> Phage Phi X 174</p>
−
<p><strong>Based on:</strong> <a href="https://doi.org/10.1038/nchembio.2233">Piraner, Dan I., et al. <cite>Nature Chemical Biology</cite> 13.1 (2017): 75-80.</a></p>
+
            <p><strong>Based on:</strong> provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich</p>
−
<p><strong>Source:</strong> gBlock</p>
+
            <p><strong>Source:</strong> plasmid provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich</p>
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500004">BBa_K2500004</a></p>
+
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500006">BBa_K2500006</a></p>
−
</details>
+
        </details>
  
−
<details>
+
        <details>
−
<summary>BBa_K2500005: RBS_TlpA</summary>
+
            <summary>BBa_K2500007: RBS<sub>eng</sub>_TlpA</summary>
−
<p><strong>Design notes:</strong> Parameters influencing the final expression of the genes downstream of pTlpA include the RBS of its repressor protein TlpA. By tweaking these sequences, it should be possible to obtain a wide range of leakiness and fold changes (fold-change up to 200-fold).</p>
+
            <p><strong>Design notes:</strong> A RBS library was created using the Red Libs algorithm to find variants translating more TlpA RNA in order to create a thermoswitch tight enough to repress the premature induction of cell lysis by protein E.</p>
−
                      <figure class="RBS_TlpA">
+
            <figure class="RBS_TlpA">
−
                      <img src="https://static.igem.org/mediawiki/2017/5/57/T--ETH_Zurich--rbs_tlpA.png">
+
                <img src="https://static.igem.org/mediawiki/2017/5/57/T--ETH_Zurich--rbs_tlpA.png">
−
                      </figure>
+
            </figure>
−
<p><strong>Organism:</strong> <span class="bacterium">Salmonella typhimurium</span></p>
+
            <p><strong>Organism:</strong> <span class="bacterium">Salmonella typhimurium</span></p>
−
<p><strong>Based on:</strong> <a href="https://doi.org/10.1038/nchembio.2233">Piraner, Dan I., et al. <cite>Nature Chemical Biology</cite> 13.1 (2017): 75-80.</a></p>
+
            <p><strong>Based on:</strong> <a href="https://doi.org/10.1038/nchembio.2233">Piraner, Dan I., et al. <cite>Nature Chemical Biology</cite> 13.1 (2017): 75-80.</a></p>
−
<p><strong>Source:</strong> gBlock</p>
+
            <p><strong>Source:</strong> FIXME RedLibs + gBlock</p>
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500005">BBa_K2500005</a></p>
+
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500007">BBa_K2500007</a></p>
−
</details>
+
        </details>
  
−
<details>
 
−
<summary>BBa_K2500006: Protein E</summary>
 
−
<p><strong>Organism:</strong> phage Phi X 174</p>
 
−
<p><strong>Based on:</strong> provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich</p>
 
−
<p><strong>Source:</strong> plasmid provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich</p>
 
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500006">BBa_K2500006</a></p>
 
−
</details>
 
  
−
<details>
+
        <details>
−
<summary>BBa_K2500007: RBS<sub>eng</sub>_TlpA</summary>
+
            <summary>BBa_K2500009: RBS<sub>eng</sub>_ProteinE</summary>
−
<p><strong>Design notes:</strong> FIXME FIXME FIXME RBS info</p>
+
            <p><strong>Design notes:</strong> A RBS library was created using the Red Libs algorithm to find variants translating less protein E RNA in order to prevent premature lysis of the bacterial cells.</p>
−
<p><strong>Organism:</strong> <span class="bacterium">Salmonella typhimurium</span></p>
+
            <figure class="RBSproteinE">
−
<p><strong>Based on:</strong> <a href="https://doi.org/10.1038/nchembio.2233">Piraner, Dan I., et al. <cite>Nature Chemical Biology</cite> 13.1 (2017): 75-80.</a></p>
+
                <img src="https://static.igem.org/mediawiki/2017/7/7d/T--ETH_Zurich--RBSproteinE.png">
−
<p><strong>Source:</strong> FIXME RedLibs + gBlock</p>
+
            </figure>
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500007">BBa_K2500007</a></p>
+
            <p><strong>Organism:</strong> Phage Phi X 174</p>
−
</details>
+
            <p><strong>Based on:</strong> provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich</p>
 +
            <p><strong>Source:</strong> FIXME RedLibs + plasmid provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich</p>
 +
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500009">BBa_K2500009</a></p>
 +
        </details>
  
−
          
+
         <details>
−
<!--<details>
+
            <summary>BBa_K2500010: AND Gate A</summary>
−
<summary>BBa_K2500008: p<sub>Const</sub>_RBS<sub>eng</sub>_TlpA</summary>
+
            <p><strong>Design notes:</strong> The AND gate integrates two signals, namely the presence of high L-lactate concentrations and high bacterial cell density (qorum sensing) and regulates the effector functions of CATE. In the absence of high concentrations of L-lactate, LldR inhibitor proteins bind to the binding sites O1 and O2 surrounding the p<sub>Lux</sub> promoter leading to the formation of a DNA loop. The p<sub>Lux</sub> promoter is sequestered and inaccessible for transcriptional activation by the quorum sensing components. In design A, the distances between the intercalated promoter and the binding sites were taken from <a href="http://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a>.</p>
−
<p><strong>Design notes:</strong> FIXME FIXME FIXME RBS info</p>
+
            <figure class="A">
−
<p><strong>Organism:</strong> <span class="bacterium">Salmonella typhimurium</span></p>
+
                <img src="https://static.igem.org/mediawiki/2017/7/72/T--ETH_Zurich--ANDgateA.png" alt="FIXME">
−
<p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_J23104">BBa_J23104</a> (promoter) and <a href="https://doi.org/10.1038/nchembio.2233">Piraner, Dan I., et al. <cite>Nature Chemical Biology</cite> 13.1 (2017): 75-80.</a> (TlpA)</p>
+
            </figure>
−
<p><strong>Source:</strong> FIXME RedLibs + gBlock</p>
+
            <p><strong>Organism:</strong> <span class="bacterium">Escherichia coli, Vibrio fischeri</span></p>
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500008">BBa_K2500008</a></p>
+
            <p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a> (O1 and O2) and <a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a> (p<sub>Lux</sub>)</p>
−
</details>-->
+
            <p><strong>Source:</strong> gBlock</p>
 +
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500010">BBa_K2500010</a></p>
 +
        </details>
  
−
<details>
+
        <details>
−
<summary>BBa_K2500009: RBS<sub>eng</sub>_ProteinE</summary>
+
            <summary>BBa_K2500011: AND Gate B</summary>
−
<p><strong>Design notes:</strong> FIXME FIXME FIXME RBS info</p>
+
            <p><strong>Design notes:</strong> In design B, each binding site was duplicated in order to achieve a potential zipper mechanism and stronger inhibition due to binding more LldR inhibitor proteins.</p>
−
<p><strong>Organism:</strong> phage Phi X 174</p>
+
            <figure class="B">
−
<p><strong>Based on:</strong> provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich</p>
+
                <img src="https://static.igem.org/mediawiki/2017/6/6a/T--ETH_Zurich--ANDgateB.png" alt="FIXME">
−
<p><strong>Source:</strong> FIXME RedLibs + plasmid provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich</p>
+
            </figure>
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500009">BBa_K2500009</a></p>
+
            <p><strong>Organism:</strong> <span class="bacterium">Escherichia coli, Vibrio fischeri</span></p>
−
</details>
+
            <p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a> (O1 and O2) and <a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a> (p<sub>Lux</sub>)</p>
 +
            <p><strong>Source:</strong> gBlock</p>
 +
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500011">BBa_K2500011</a></p>
 +
        </details>
  
 +
        <details>
 +
            <summary>BBa_K2500012: AND Gate C</summary>
 +
            <p><strong>Design notes:</strong> In design C, an artificial spacer was embedded between the pLux promoter and the O2 binding site in order to influence the looping dynamics.</p>
 +
            <figure class="C">
 +
                <img src="https://static.igem.org/mediawiki/2017/c/c2/T--ETH_Zurich--ANDgateC.png" alt="FIXME">
 +
            </figure>
 +
            <p><strong>Organism:</strong> <span class="bacterium">Escherichia coli, Vibrio fischeri</span></p>
 +
            <p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a> (O1 and O2) and <a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a> (p<sub>Lux</sub>)</p>
 +
            <p><strong>Source:</strong> gBlock</p>
 +
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500012">BBa_K2500012</a></p>
 +
        </details>
  
−
<details>
+
        <details>
−
<summary>BBa_K2500010: AND Gate A</summary>
+
            <summary>BBa_K2500013: p<sub>Const1</sub>_RBS_LldP/LldR_p<sub>Const2</sub>_RBS_LuxR</summary>
−
<p><strong>Design notes:</strong> In the absence of high concentrations of L-lactate, LldR inhibitor proteins bind to the binding sites O1 and O2 surrounding the p<sub>Lux</sub> promoter leading to the formation of a DNA loop. The p<sub>Lux</sub> promoter is sequestered and inaccessible for transcription. In design A, the distances between the intercalated promoter and the binding sites were taken from <a href="http://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a>.</p>
+
            <p><strong>Design notes:</strong>This expression cassette regulates the two inputs relayed to our AND gate, namely lactate and quorum sensing. LLdp is a L-lactate permease which transports lactate across the bacterial cell membrane. High lactate concentrations inhibit the LldR repressor proteins which under low-lactate conditions prevent access to the pLux quorum sensing promoter by LuxR-AHL complexes.</p>
−
<figure class="A">
+
−
<img src="https://static.igem.org/mediawiki/2017/7/72/T--ETH_Zurich--ANDgateA.png" alt="FIXME">
+
−
</figure>
+
−
<p><strong>Organism:</strong> <span class="bacterium">FIXME FIXME FIXME</span></p>
+
−
<p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a> (O1 and O2) and <a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a> (p<sub>Lux</sub>)</p>
+
−
<p><strong>Source:</strong> gBlock</p>
+
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500010">BBa_K2500010</a></p>
+
−
</details>
+
  
−
<details>
+
            <p>Higher bacterial cell densities result in a higher density of AHL communication molecules. AHL molecules diffuse across bacterial cell membranes, bind the transcription activator LuxR and activate the transcription of genes downstream of the pLux promoter, which is only accessible under high lactate levels as described above.</p>
−
<summary>BBa_K2500011: AND Gate B</summary>
+
            <figure class="cassette">
−
<p><strong>Design notes:</strong> In design B, each binding site was duplicated in order to achieve a potential zipper mechanism and stronger inhibition due to binding more LldR inhibitor proteins.</p>
+
                <img src="https://static.igem.org/mediawiki/2017/1/13/T--ETH_Zurich--expressioncassette.png">
−
<figure class="B">
+
            </figure>
−
<img src="https://static.igem.org/mediawiki/2017/6/6a/T--ETH_Zurich--ANDgateB.png" alt="FIXME">
+
            <p><strong>Organism:</strong> <span class="bacterium">Escherichia coli, Vibrio fischeri</span></p>
−
</figure>
+
            <p><strong>Based on:</strong> FIXME <a href="http://parts.igem.org/Part:BBa_J23118">BBa_J23118</a> (p<sub>Const1</sub>), <a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a> (p<sub>Const2</sub>), <a href="http://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a> (LldP/LldR) and <a href="http://parts.igem.org/Part:BBa_C0062">BBa_C0062</a> (LuxR)</p>
−
<p><strong>Organism:</strong> <span class="bacterium">FIXME FIXME FIXME</span></p>
+
            <p><strong>Source:</strong> FIXME</p>
−
<p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a> (O1 and O2) and <a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a> (p<sub>Lux</sub>)</p>
+
            <p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500013">BBa_K2500013</a></p>
−
<p><strong>Source:</strong> gBlock</p>
+
        </details>
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500011">BBa_K2500011</a></p>
+
    </div>
−
</details>
+
−
 
+
−
<details>
+
−
<summary>BBa_K2500012: AND Gate C</summary>
+
−
<p><strong>Design notes:</strong> In design C, an artificial spacer was embedded between the pLux promoter and the O2 binding site in order to influence the looping dynamics.</p>
+
−
<figure class="C">
+
−
<img src="https://static.igem.org/mediawiki/2017/c/c2/T--ETH_Zurich--ANDgateC.png" alt="FIXME">
+
−
</figure>
+
−
<p><strong>Organism:</strong> <span class="bacterium">FIXME FIXME FIXME</span></p>
+
−
<p><strong>Based on:</strong> <a href="http://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a> (O1 and O2) and <a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a> (p<sub>Lux</sub>)</p>
+
−
<p><strong>Source:</strong> gBlock</p>
+
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500012">BBa_K2500012</a></p>
+
−
</details>
+
−
 
+
−
<details>
+
−
<summary>BBa_K2500013: p<sub>Const1</sub>_RBS_LldP/LldR_p<sub>Const2</sub>_RBS_LuxR</summary>
+
−
<p><strong>Design notes:</strong>FIXME FIXME FIXME</p>
+
−
<p><strong>Organism:</strong> <span class="bacterium">FIXME FIXME FIXME</span></p>
+
−
<p><strong>Based on:</strong> FIXME <a href="http://parts.igem.org/Part:BBa_J23118">BBa_J23118</a> (p<sub>Const1</sub>), <a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a> (p<sub>Const2</sub>), <a href="http://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a> (LldP/LldR) and <a href="http://parts.igem.org/Part:BBa_C0062">BBa_C0062</a> (LuxR)</p>
+
−
<p><strong>Source:</strong> FIXME</p>
+
−
<p><strong>Registry:</strong> <a href="http://parts.igem.org/Part:BBa_K2500013">BBa_K2500013</a></p>
+
−
</details>
+
−
 
+
−
</div>
+
 
</section>
 
</section>
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</main>
 
</main>
 
</html>
 
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{{ETH_Zurich/Footer_N}}
 
{{ETH_Zurich/Footer_N}}

Latest revision as of 01:50, 2 November 2017

Basic Parts

FIXME

You can read details about the design of our Basic Parts here. To check out all the parts that we created, visit Parts.

Overview

Part Description BioBrick
Best Basic Part:
AND Gate B		 Synthetic promoter responsive to LldR and luxR BBa_K2500011
Bacterioferritin Heme-deletion mutant of bacterial iron storage protein functioning as an MRI contrast agent BBa_K2500000
Azurin Redox protein originating from P. aeruginosa with cytotoxic activity BBa_K2500001
p28 Effector domain of azurin BBa_K2500002
PTlpA Temperature-responsive promoter optimized for slight activation above 37°C and full activation at 45 °C BBa_K2500003
TlpA Temperature-dependent transcriptional repressor of pTlpa BBa_K2500004
RBS_TlpA Temperature-dependent transcriptional repressor of pTlpa and synthetic RBS BBa_K2500005
Protein E Bacteria-lysing protein encoded by phage Phi X 17 BBa_K2500006
RBSeng_TlpA Temperature-dependent transcriptional repressor of pTlpa with engineered RBS BBa_K2500007
RBSeng_ProteinE Bacteria-lysing protein encoded by phage Phi X 174 with engineered RBS BBa_K2500009
AND Gate A Synthetic promoter responsive to LldR and luxR BBa_K2500010
AND Gate C Synthetic promoter responsive to LldR and luxR BBa_K2500012
PConst_RBS_LldP/
LldR_PConst_RBS_LuxR		 Expression cassette consisting of LuxR and LldP/LldR BBa_K2500013

Design

All parts were codon-optimized for expression in E. coli Nissle with Geneious and modified to remove forbidden restriction sites.

BBa_K2500000: Bacterioferritin

Design notes: Bacterioferritins are bacterial iron storage proteins. It has been shown that overexpression of bacterioferritin in E. coli Nissle 1917 can lead to a visible contrast change in MRI, which allows for visualization of the bacteria. We used a heme-deletion mutant as it does not decrease contrast change.

Organism: Escherichia coli Nissle

Based on: BBa_K1438001

Source: gBlock

Registry: BBa_K2500000

BBa_K2500001: Azurin

Design notes: Azurin is a redox protein with the ability to induce apoptosis in mammalian cells upon internalization. We removed the native signal peptide which is part of the pre-protein form of azurin as no targeting within the bacterial host cells is required.

Organism: Pseudomonas aeruginosa

Based on: BBa_K835004

Source: gBlock

Registry: BBa_K2500001

BBa_K2500002: p28

Design notes: The effector domain of azurin comprises amino acids 50 to 77. We extracted the sequence, introduced a start codon (ATG) at the beginning and two stop codons (TAA TAA) at the end. By encoding only the effector, we were aiming at achieving faster expression levels and a higher cytotoxic load carried by the bacteria.

Organism: Pseudomonas aeruginosa

Based on: BBa_K2500001

Source: gBlock

Registry: BBa_K2500002

BBa_K2500003: pTlpA

Design notes: The pTlpA promoter regulates the lysis step of CATE. The promoter is bound and inhibited by TlpA repressor proteins below 37 °C, thereby blocking the release of the cytotoxic compound at body temperature. To fully activate the promoter and thus cell lysis, the desired area is heated to 45 °C with focused ultrasound.

Organism: Salmonella typhimurium

Based on: Piraner, Dan I., et al. Nature Chemical Biology 13.1 (2017): 75-80.

Source: oligonucleotide sequence

Registry: BBa_K2500003

BBa_K2500004: TlpA

Design notes: TlpA is a constitutively expressed repressor protein which inhibits transcription of genes downstream of the TlpA promoter by binding to it. The binding to the promoter is reversibly deactivated by temperatures above 37 °C with full deactivation at 45 °C.

Organism: Salmonella typhimurium

Based on: Piraner, Dan I., et al. Nature Chemical Biology 13.1 (2017): 75-80.

Source: gBlock

Registry: BBa_K2500004

BBa_K2500005: RBS_TlpA

Design notes: Parameters influencing the final expression levels of the genes downstream of pTlpA include the RBS of its repressor protein TlpA. By tweaking these sequences, it should be possible to obtain a wide range of leakiness and fold changes (fold-change up to 200-fold).

Organism: Salmonella typhimurium

Based on: Piraner, Dan I., et al. Nature Chemical Biology 13.1 (2017): 75-80.

Source: gBlock

Registry: BBa_K2500005

BBa_K2500006: Protein E

Design notes: Protein E is a lysis protein originally encoded by the bacteriophage Phi X 147. It is now generally accepted that the most probable cellular target of protein E is the enzyme translocase I, encoded by the mraY gene. Translocase I is required for cell wall biosynthesis and its inhibition results to cell lysis.

Organism: Phage Phi X 174

Based on: provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich

Source: plasmid provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich

Registry: BBa_K2500006

BBa_K2500007: RBSeng_TlpA

Design notes: A RBS library was created using the Red Libs algorithm to find variants translating more TlpA RNA in order to create a thermoswitch tight enough to repress the premature induction of cell lysis by protein E.

Organism: Salmonella typhimurium

Based on: Piraner, Dan I., et al. Nature Chemical Biology 13.1 (2017): 75-80.

Source: FIXME RedLibs + gBlock

Registry: BBa_K2500007

BBa_K2500009: RBSeng_ProteinE

Design notes: A RBS library was created using the Red Libs algorithm to find variants translating less protein E RNA in order to prevent premature lysis of the bacterial cells.

Organism: Phage Phi X 174

Based on: provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich

Source: FIXME RedLibs + plasmid provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich

Registry: BBa_K2500009

BBa_K2500010: AND Gate A

Design notes: The AND gate integrates two signals, namely the presence of high L-lactate concentrations and high bacterial cell density (qorum sensing) and regulates the effector functions of CATE. In the absence of high concentrations of L-lactate, LldR inhibitor proteins bind to the binding sites O1 and O2 surrounding the pLux promoter leading to the formation of a DNA loop. The pLux promoter is sequestered and inaccessible for transcriptional activation by the quorum sensing components. In design A, the distances between the intercalated promoter and the binding sites were taken from BBa_K1847007.

FIXME

Organism: Escherichia coli, Vibrio fischeri

Based on: BBa_K1847007 (O1 and O2) and BBa_R0062 (pLux)

Source: gBlock

Registry: BBa_K2500010

BBa_K2500011: AND Gate B

Design notes: In design B, each binding site was duplicated in order to achieve a potential zipper mechanism and stronger inhibition due to binding more LldR inhibitor proteins.

FIXME

Organism: Escherichia coli, Vibrio fischeri

Based on: BBa_K1847007 (O1 and O2) and BBa_R0062 (pLux)

Source: gBlock

Registry: BBa_K2500011

BBa_K2500012: AND Gate C

Design notes: In design C, an artificial spacer was embedded between the pLux promoter and the O2 binding site in order to influence the looping dynamics.

FIXME

Organism: Escherichia coli, Vibrio fischeri

Based on: BBa_K1847007 (O1 and O2) and BBa_R0062 (pLux)

Source: gBlock

Registry: BBa_K2500012

BBa_K2500013: pConst1_RBS_LldP/LldR_pConst2_RBS_LuxR

Design notes:This expression cassette regulates the two inputs relayed to our AND gate, namely lactate and quorum sensing. LLdp is a L-lactate permease which transports lactate across the bacterial cell membrane. High lactate concentrations inhibit the LldR repressor proteins which under low-lactate conditions prevent access to the pLux quorum sensing promoter by LuxR-AHL complexes.

Higher bacterial cell densities result in a higher density of AHL communication molecules. AHL molecules diffuse across bacterial cell membranes, bind the transcription activator LuxR and activate the transcription of genes downstream of the pLux promoter, which is only accessible under high lactate levels as described above.

Organism: Escherichia coli, Vibrio fischeri

Based on: FIXME BBa_J23118 (pConst1), BBa_J23100 (pConst2), BBa_K1847007 (LldP/LldR) and BBa_C0062 (LuxR)

Source: FIXME

Registry: BBa_K2500013