Difference between revisions of "Team:ETH Zurich/Experiments"

 
(32 intermediate revisions by 3 users not shown)
Line 9: Line 9:
 
         --><li class="outline_item"><a href="#trans">Competent Cells</a></li><!--
 
         --><li class="outline_item"><a href="#trans">Competent Cells</a></li><!--
 
         --><li class="outline_item"><a href="#buffer">Buffers & Media</a></li><!--
 
         --><li class="outline_item"><a href="#buffer">Buffers & Media</a></li><!--
 +
        --><li class="outline_item"><a href="#Measurements">Measurements</a></li><!--
 
         -->
 
         -->
 
         </ul>
 
         </ul>
Line 33: Line 34:
 
<div class="sec white">
 
<div class="sec white">
 
<div>
 
<div>
−
<h3>New England Biolabs Phusion High-Fidelity DNA Polymerase</h3>
+
<h3><i style="color:#1f407a;">New England Biolabs Phusion High-Fidelity DNA Polymerase</i></h3>
 
   <h4>Composition:</h4>
 
   <h4>Composition:</h4>
 
         <table>
 
         <table>
Line 98: Line 99:
 
             <tr>
 
             <tr>
 
                  
 
                  
−
                 <td align="center"><strong>Temperatur <br />
+
                 <td align="center"><strong>Temperature <br />
 
                 </strong></td>
 
                 </strong></td>
 
                 <td align="center"><strong>Time<br />
 
                 <td align="center"><strong>Time<br />
Line 126: Line 127:
 
     </table>
 
     </table>
  
−
<p>The appropriate annealing temperature was calculated from NEB's <a href="http://tmcalculator.neb.com/#!/" ><i>Tm Calculator</i></a>
+
<p><br>The appropriate annealing temperature was calculated from NEB's <a href="http://tmcalculator.neb.com/#!/" ><i>Tm Calculator</i></a>
 
</p>
 
</p>
 
</div>
 
</div>
Line 188: Line 189:
 
             <tr>
 
             <tr>
 
                  
 
                  
−
                 <td align="center"><strong>Temperatur <br />
+
                 <td align="center"><strong>Temperature <br />
 
                 </strong></td>
 
                 </strong></td>
 
                 <td align="center"><strong>Time<br />
 
                 <td align="center"><strong>Time<br />
Line 215: Line 216:
 
         </tbody>
 
         </tbody>
 
     </table>
 
     </table>
−
<p> Kapa Hifi Hotstart has similar annealing temperatures as Phusion DNA polymerase, even slightly higher. Only in very few cases a lower annealing temperature was found to be better.</p>
+
<p><br> Kapa Hifi Hotstart has similar annealing temperatures as Phusion DNA polymerase, even slightly higher. Only in very few cases a lower annealing temperature was found to be better.</p>
 
</div>
 
</div>
 
</div>
 
</div>
Line 224: Line 225:
 
<div class="sec white" id="PCR">
 
<div class="sec white" id="PCR">
 
<div>
 
<div>
−
<h2>Polymerase Chain Reaction - for Analysis</h2>
+
<h2>Polymerase Chain Reaction - Colony PCR</h2>
 +
<p>For screening a large number of clones, single colonies were resuspended in 20 &micro;l LB medium of which 1 &micro;l was used as template for the PCR.</p>
 
</div>
 
</div>
 
</div>
 
</div>
Line 296: Line 298:
 
             <tr>
 
             <tr>
 
                  
 
                  
−
                 <td align="center"><strong>Temperatur <br />
+
                 <td align="center"><strong>Temperature <br />
 
                 </strong></td>
 
                 </strong></td>
 
                 <td align="center"><strong>Time<br />
 
                 <td align="center"><strong>Time<br />
Line 330: Line 332:
 
<div class="sec white">
 
<div class="sec white">
 
<div>
 
<div>
−
<h3>New England Biolabs <i>Taq</i> DNA Polymerase</h3>
+
<h3>New England Biolabs Taq DNA Polymerase</h3>
 
   <h4>Composition:</h4>
 
   <h4>Composition:</h4>
 
         <table>
 
         <table>
Line 386: Line 388:
 
         </tbody>
 
         </tbody>
 
     </table>
 
     </table>
−
    Create a mastermix, aliquote into PCR tubes and add 1 &micro;l the template (resuspended colony from plate in 20 &micro;l LB medium).
+
 
      
 
      
 
<h4>Thermocycling Conditions:</h4>
 
<h4>Thermocycling Conditions:</h4>
Line 393: Line 395:
 
             <tr>
 
             <tr>
 
                  
 
                  
−
                 <td align="center"><strong>Temperatur <br />
+
                 <td align="center"><strong>Temperature <br />
 
                 </strong></td>
 
                 </strong></td>
 
                 <td align="center"><strong>Time<br />
 
                 <td align="center"><strong>Time<br />
Line 420: Line 422:
 
         </tbody>
 
         </tbody>
 
     </table>
 
     </table>
−
     <p>The appropriate annealing temperature was calculated from NEB's <a href="http://tmcalculator.neb.com/#!/" ><i>Tm Calculator</i></a>
+
     <p><br>The appropriate annealing temperature was calculated from NEB's <a href="http://tmcalculator.neb.com/#!/" ><i>Tm Calculator</i></a>
 
</p>
 
</p>
  
Line 430: Line 432:
 
<div class="sec white">
 
<div class="sec white">
 
<div>
 
<div>
−
<h3>Site-Directed Mutagenesis (QuickChange)</h3>
+
<h2>Site-Directed Mutagenesis (QuickChange)</h2>
 
    
 
    
 
         <table>
 
         <table>
Line 481: Line 483:
 
             <tr>
 
             <tr>
 
                  
 
                  
−
                 <td align="center"><strong>Temperatur <br />
+
                 <td align="center"><strong>Temperature <br />
 
                 </strong></td>
 
                 </strong></td>
 
                 <td align="center"><strong>Time<br />
 
                 <td align="center"><strong>Time<br />
Line 508: Line 510:
 
         </tbody>
 
         </tbody>
 
     </table>
 
     </table>
−
<p> Primers were designed according to the guidlines of <a href="http://openwetware.org/wiki/Richard_Lab" >The Richard Lab</a> <sup><a href=#richard class="cit">1</a>
+
<p> <br>Primers were designed according to the guidlines of <a href="http://openwetware.org/wiki/Richard_Lab" >The Richard Lab</a> <sup><a href=#richard class="cit">1</a>
 
<ul>
 
<ul>
 
     <li> The targeted mutation should be included into both primers.
 
     <li> The targeted mutation should be included into both primers.
−
<li> The mutation can be as close as 4 bases from the 5-terminus.
+
<li> The mutation can be as close as 4 bases from the 5'-terminus.
−
<li> The mutation should be at least 8 bases from the 3-terminus.
+
<li> The mutation should be at least 8 bases from the 3'-terminus.
 
<li> At least eight non-overlapping bases should be introduced at the 3-end of each primer.
 
<li> At least eight non-overlapping bases should be introduced at the 3-end of each primer.
 
<li> At least one G or C should be at the end of each primer.
 
<li> At least one G or C should be at the end of each primer.
Line 518: Line 520:
  
 
</ul>
 
</ul>
−
The resulting PCR product has to be purified (e.x. Agencourt AMPure XP) and digested with DpnI (NEB) for 4 hours and gel-purified. In case Phusion polymerase is used, DpnI can directly be added to the PCR. The product can then immediately be used for transformation. With proper removal of template plasmid DNA the efficiency of exchanged bases is very high.
+
<p>The resulting PCR product has to be purified (e.x. Agencourt AMPure XP) and digested with DpnI (NEB) for 4 hours and gel-purified. In case Phusion polymerase is used, DpnI can directly be added to the PCR. The product can then immediately be used for transformation. In order to obtain high base exchange efficiency, the template need to be removed from the reaction thoroughly.
 
</p>
 
</p>
 
</div>
 
</div>
Line 524: Line 526:
  
  
−
<div class="sec white" id="assembly">
+
<div class="sec light_grey" id="assembly">
 
<div>
 
<div>
−
<h2>Isothermal "Gibson" Assembly</h2>
+
<h2>Isothermal "Gibson" Assembly<sup><a href=#gibson class="cit">2</a></h2>
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<div class="sec white">
+
<div class="sec light_grey">
 
<div>
 
<div>
 
<h3>Recipe for Ready-to-Use Isothermal Assembly Mixes</h3>
 
<h3>Recipe for Ready-to-Use Isothermal Assembly Mixes</h3>
Line 549: Line 551:
 
     </ul>
 
     </ul>
  
−
    This buffer can be aliquoted and stored at -20 &deg;C.
+
  <p>  <br>This buffer can be aliquoted and stored at -20 &deg;C.</p>
  
  
Line 562: Line 564:
  
 
</ul>
 
</ul>
−
The master mix is devided into aliquots of 15 &micro;l and stored at -20 &deg;C.  
+
<p><br>The master mix is devided into aliquots of 15 &micro;l and stored at -20 &deg;C. </p>
  
  
Line 569: Line 571:
 
</div>
 
</div>
  
−
<div class="sec white">
+
<div class="sec light_grey">
 
<div>
 
<div>
 
<h3>Protocol for Isothermal Assembly</h3>
 
<h3>Protocol for Isothermal Assembly</h3>
Line 576: Line 578:
 
     <li>15 &micro;l aliquot of master mix
 
     <li>15 &micro;l aliquot of master mix
 
     <li>0.02-0.5 pmol DNA in total for 2-3 fragments<br>
 
     <li>0.02-0.5 pmol DNA in total for 2-3 fragments<br>
−
            or
+
        <ul>  or </ul>
 
     <li>0.2-1 pmol DNA in total for 4-6 fragments
 
     <li>0.2-1 pmol DNA in total for 4-6 fragments
 
         <li>Fill up with water to 20 &micro;l     
 
         <li>Fill up with water to 20 &micro;l     
Line 587: Line 589:
 
             </ul>
 
             </ul>
  
−
            The assembled mix is then incubated for 60 minutes at 50 &deg;C. It can be directly transformed with chemically competent cell (volume of assembly reaction not exceeding 10% of the volume of the competent cells). Otherwise, the reaction mix is purified and desalted (e.x. Agencourt AMPure XP) and a fraction of it transformed with electrocompetent cells.
+
          <p> <br>The assembled mix is then incubated for 60 minutes at 50 &deg;C. It can be directly transformed with chemically competent cell (volume of assembly reaction not exceeding 10% of the volume of the competent cells). Otherwise, the reaction mix is purified and desalted (e.x. Agencourt AMPure XP) and a fraction of it transformed with electrocompetent cells.</p>
 
</div>
 
</div>
 
</div>
 
</div>
Line 606: Line 608:
 
     <li>Cool the culture on ice, transfer the cells into cetrifugation tubes and harvest them by centrifugation for 5 min (4000xg, 4&deg;C)
 
     <li>Cool the culture on ice, transfer the cells into cetrifugation tubes and harvest them by centrifugation for 5 min (4000xg, 4&deg;C)
 
     <li>Carfully discard supernatant, keep cells always on ice.
 
     <li>Carfully discard supernatant, keep cells always on ice.
−
     <li>Resuspend cells in 30 ml cold <a href="#TFB1" class="cit">TFB1</a> and incubate on ice for 90 minutes.
+
     <li>Resuspend cells in 30 ml cold <a href="#TFB1">TFB1</a> and incubate on ice for 90 minutes.
−
     <li>
+
     <li>Centrifuge for 5 min (4000xg, 4&deg;C)
 +
    <li>Carfully discard supernatant, keep cells always on ice.
 +
    <li>Resuspend the cells in 4 ml ice-cold <a href="#TFB2">TFB2</a> buffer.
 +
    <li>Prepare aliquots of 100 &micro;l in pre-cooled, sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at -80&deg; C
 +
        </ul>
 +
   
 +
    <h4>Transformation Protocol for Chemically Competent Cells</h4>
 +
    <ul>
 +
        <li>Thaw the competent cells on ice
 +
        <li>Use 50 ul of the competent cells for one transformation, do not add more than 5 ul of DNA to the cells (10% of the volume of the competent cells)
 +
        <li>Incubate them for 20 min on ice
 +
        <li>Incubate them for 90 s at 42&deg; C and add afterwards 500 &micro;l of <a href="#SOC">SOC</a>
 +
        <li>Let them regenerate for 1 h at 37&deg; C with shaking
 +
        <li>Plate a part of the culture according to your expectations on LB-agar plates with the approbriate antibiotic.
 +
    </ul>
 +
</div>
 +
</div>
  
  
  
  
 +
<div class="sec white">
 +
<div>
 +
<h3>Preparation of Electrocompetent Cells</h3>
 +
<ul>
 +
    <li>Inoculate 600 ml of prewarmed LB medium with 6 ml overnight culture and grow the bacteria to an OD<sub>600</sub> of 0.4.
 +
    <li>Cool the culture on ice fpr 30 min, transfer 300 ml of the culture into 50 ml tubes and centrifuge them for 10 min (3000xg, 4&deg;C). Discard supernatant and add the rest of the culture. Again discard supernatant.
 +
    <li>Carfully resuspend cells in ice-cold, autoclaved and deionized water and fill up to 50 ml
 +
    <li>Centrifuge again for 10 min (3000xg, 4&deg;C) and repeat wash step from above
 +
    <li>Carfully resuspend cells in ice-cold 10% glycerol (working always on ice)
 +
    <li>Centrifuge for 10 min (3000xg, 4&deg;C)
 +
    <li>Discard supernatant
 +
    <li>Resuspend the cells in the remaining glycerol solution.
 +
    <li>Prepare aliquots of 35 &micro;l in pre-cooled, sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at -80&deg; C
 +
        </ul>
  
 +
<h4>Transformation Protocol for Electrocompetent Cells</h4>
 +
    <ul>
 +
        <li>Thaw the electrocompetent cells on ice. Pre-chill also the electroporation cuvette.
 +
        <li>Add DNA to the cells. Attention: the DNA should contain as little ions as possible. Purify or dilute reactions containing salt.
 +
        <li>Transfer the bacteria/DNA mix into a pre-chilled electroporation cuvette and electroporate them.
 +
        <li>Add IMMEDIATELY 500 &micro;l of <a href="#SOC">SOC</a> and transfer them back into the microcentrifuge tube.
 +
        <li>Let them regenerate for 1 h at 37&deg; C with sufficient shaking.
 +
        <li>Plate a part of the transformation mix according to your expectations on LB-agar plates with the approbriate antibiotic.
 +
    </ul>
  
−
<a name="TFB1" class></a>
+
</div>
 +
</div>
  
  
−
</body>
+
<div class="sec light_grey">
−
</html>
+
<div>
  
−
{{:Template:ETH_Zurich/footer}}
+
<h2>Buffer and Medium</h2>
  
 +
<div class="sec light_grey" id="TFB1">
 +
    <h3>TFB1</h3>
 +
       
  
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>Concentration</strong></td>
 +
                <td><strong>Amount per 100 ml</strong></td>
 +
                </tr>
 +
           
 +
            <tr>
 +
                <td>RbCl</td>
 +
                <td>100 mM</td>
 +
              <td>1.21 g</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>MnCl<sub>2</sub> &middot; 4 H<sub>2</sub>O</td>
 +
                <td>50 mM</td>
 +
                <td>0.99 g</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>Potassium acetate &middot; 4 H<sub>2</sub>O</td>
 +
                <td>30 mM</td>
 +
                <td>0.29 g</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>CaCl<sub>2</sub> &middot; 2 H<sub>2</sub>O</td>
 +
                <td>10 mM</td>
 +
                <td>0.147 g</td>
 +
             
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>Glycerol</td>
 +
                <td>15 %</td>
 +
                <td>11.9 g</td>
 +
               
 +
            </tr>
 +
         
  
  
 +
        </tbody>
 +
    </table>
  
 +
    <p> <br>Adjust pH to 5.5 and sterilize by filtration</p>
 +
    </div>
 +
</div>
  
 +
<div class="sec light_grey" id="TFB2">
 +
    <h3>TFB2</h3>
 +
       
  
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>Concentration</strong></td>
 +
                <td><strong>Amount per 100 ml</strong></td>
 +
                </tr>
 +
           
 +
            <tr>
 +
                <td>MOPS</td>
 +
                <td>100 mM</td>
 +
              <td>0.0047 g</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>RbCl</td>
 +
                <td>50 mM</td>
 +
                <td>0.0027 g</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>CaCl<sub>2</sub> &middot; 2 H<sub>2</sub>O</td>
 +
                <td>75 mM</td>
 +
                <td>0.024 g</td>
 +
             
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>Glycerol</td>
 +
                <td>15 %</td>
 +
                <td>0.27g</td>
 +
               
 +
            </tr>
 +
         
  
  
 +
        </tbody>
 +
    </table>
  
−
<h3>Nitric Oxide</h3>
+
     <p> <br>Adjust pH to 6.5 and sterilize by filtration</p>
−
<div class="image_box" style="max-width: 400px;">
+
    </div>
−
    <a href="https://2016.igem.org/File:T--ETH_Zurich--NorR_draft_vector.svg">
+
−
        <img src="https://static.igem.org/mediawiki/2016/c/c0/T--ETH_Zurich--NorR_draft_vector.svg">
+
−
    </a>
+
−
     <p><b>Figure 1:</b> NorR constitutively binds to DNA. Only when NO<sup>&#8226;</sup> is present it activates the transcription of the gene under control of the norVW promoter (adapted from <a href="#green2014transcriptional" >Green <i>et al.</i></a>) </p>
+
−
</div><p>
+
−
Beside the penetration of immunogenic antigens across the epithelial layer, there is also non-normal leakage of inflammation markers into the gut lumen. One of these molecules is nitric oxide (NO<sup>&#8226;</sup>, <i>t</i><sub>1/2</sub> < 6 seconds<sup><a href="#kochar2011nitric" class="cit">7</a></sup>) and is one of the molecules we are going to sense with our system. The sensing of NO<sup>&#8226;</sup> with <i>E. coli</i> has already been described by Archer <i>et al.</i><sup><a href="#archer2012engineered" class="cit">8</a></sup> in 2012. This work provides us with the relevant genetic elements and helps us to design this system for our purpose. Additionally, they present their system as a rapid detection system for IBD related disease flare-ups which would allow for an immediate intervention. <br>
+
−
NorR is capable of binding NO<sup>&#8226;</sup> with its mononuclear non-heme iron center. While other sensor proteins are not only specific for NO<sup>&#8226;</sup> but also for other NO<sub>x</sub> species, NorR binds specifically the NO<sup>&#8226;</sup> radical. NorR is constitutively bound as a hexamer upstream of the norVW promoter but inhibiting transcription in absence of NO<sup>&#8226;</sup>. Once NO<sup>&#8226;</sup> binds to NorR, its ATPase activity is triggered and provides energy to form a productive interaction with the &#963;<sup>54</sup> - RNA polymerase holoenzyme<sup><a href="#green2014transcriptional" class="cit">9</a></sup>.
+
−
</p>
+
−
</div>
+
−
</div>
+
  
−
<div class="sec light_grey">
 
−
<div>
 
−
<h3>N-Acyl Homoserine Lactones</h3>
 
−
<div class="image_box" style="max-width: 400px;">
 
−
    <a href="https://static.igem.org/mediawiki/2016/6/6d/T--ETH_Zurich--TraR_colored.jpg">
 
−
        <img src="https://static.igem.org/mediawiki/2016/6/6d/T--ETH_Zurich--TraR_colored.jpg">
 
−
    </a>
 
−
    <p><b>Figure 2:</b> DNA- and 3-oxo-C6-HSL bound, dimeric form of TraR, a close homolog of EsaR (PDB: <a href="http://www.rcsb.org/pdb/explore.do?structureId=1l3l" >1L3L</a>, edited with UCSF Chimera<sup><a href="#chimera" class="cit">1</a></sup>) </p>
 
−
</div><p>
 
−
In addition to a general inflammation marker we want to sense molecules secreted by the microbiota in order to identify the bacteria. One well-known class of molecules secreted by many bacterial species belongs to the quorum sensing (QS) system. QS molecules act as bacterial hormones among and between species which control for example the formation of biofilms and growth behaviour. Furthermore, QS molecules can alter the microbiota's composition<sup><a href="#thompson2015manipulation" class="cit">1</a></sup>. The best known subclass of QS molecules are the N-acyl homoserine lactones (AHL) which will be identified by our living biosensor. <br>
 
−
One of the AHLs to be found upregulated in IBD<sup><a href="#landman2013sa1804" class="cit">1</a></sup> is 3-hydroxy-hexanoyl-HSL (3-OH-C6-HSL). A well characterized regulatory protein that senses a very similar HSL (3-oxo-C6-HSL) is EsaR from <i>Erwinia stewartii</i> that was used by a <a href="https://2015.igem.org/Team:Manchester-Graz/Project/Vectordesign" >previous iGEM team</a>. The special feature of EsaR is its regulatory behaviour: while most HSL-responsive elements are inducible activators, EsaR is a repressor that dissociates from the DNA in presence of HSL. This is important for our circuit as a repressor is thought to be less leaky than an activator. <br>
 
−
As our target HSL is not the natural ligand for EsaR, we applied a directed evolution strategy to change its specificity.
 
  
−
</p>
+
<div class="sec light_grey" id="LB Medium">
−
</div>
+
    <h3>LB Medium</h3>
−
</div>
+
       
  
−
<div class="sec dark_grey" id="learning">
+
        <table>
−
<div>
+
        <tbody>
−
<h2>Associative Learning Circuit</h2>
+
            <tr>
−
<h3>Overview</h3>
+
                <td><strong>Component</strong></td>
−
<p>
+
                <td><strong>Concentration</strong></td>
−
To serve as a diagnostics and research tool, our system should not only be able to sense a single molecule alone but should associate an inflammation marker - in our case NO<sup>&#8226;</sup> - with a potential trigger of the inflammation itself. Thus, we implemented an associative learning circuit that allows for the detection of the temporal and spatial presence of two markers. <br>
+
                <td><strong>Amount per 1l</strong></td>
−
Nitric oxide and 3-OH-C6-HSL are only two possbile markers of IBD. There exist many more that are definitively worth to be further investigated and are ideally observed in parallel. This is why we extended the AND-gate by a learning component. While the number of distinguistable reporters (e.g. fluorophores) is limited, our system allows for simultanious observation of a multitude of parallel measured markers. Our <i>Pavlov's Coli</i> learn the occurence of the presence of two markers and store this information in their DNA until readout. <br>
+
                </tr>
−
We designed our system in a way that allows fast and easy demultiplexing of a complex mixture of different reporter strains. If the reporter strains encounter again the with inflammation associated marker, they generate an easily observable output: fluorescence. This was achieved by integrating a second AND-gate that relies on the successful learning process. <br>
+
           
−
</p>
+
            <tr>
−
</div>
+
                <td>Bacto-tryptone</td>
−
</div>
+
                <td>1%</td>
 +
              <td>10g</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>Yeast Extract</td>
 +
                <td>0.5%</td>
 +
                <td>5g</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>NaCl</td>
 +
                <td>1%</td>
 +
                <td>10g</td>
 +
             
 +
            </tr>
 +
         
  
−
<div class="sec white">
 
−
<div>
 
−
<h3>Biological Implementation: Recombinase</h3>
 
−
</div>
 
−
</div>
 
  
−
<div class="sec white three_columns">
+
        </tbody>
−
<div>
+
    </table>
  
−
<div>
+
    <p> <br>Sterilized by autoclaving</p>
−
<h4>Sensor AND-gate:</h4><br>
+
    </div>
−
<p>
+
−
At an inflammation spot, nitric oxide activates NorR and triggers the transcription of the <i>bxb1</i> integrase gene. The transcription can only proceed if 3-OH-C6-HSL is present. The HSL lets the repressor EsaR dissociate from the regulatory element (esaBox) on the DNA and thus annihiliates its roadblock activity.<br>
+
−
</p>
+
−
</div>
+
  
−
<div>
+
<div class="sec light_grey" id="LB-Agar">
−
<h4>Learning:</h4><br>
+
    <h3>LB Agar</h3>
−
<p>
+
       
−
Once the <i>bxb1</i> gene is successfully transcribed and translated, Bxb1 binds to the attP and attB recombination sites flanking a constitutive promoter and inverts it. As attP and attB are destroyed through inversion, Bxb1 mediated recombination acts as a one-way switch. <br>
+
−
</p>
+
−
</div>
+
  
−
<div>
+
        <table>
−
<h4>Reporter AND-gate:</h4><br>
+
        <tbody>
−
<p>
+
            <tr>
−
The constitutive promoter, now being placed upstream of the reporter protein GFP, is further under control of another esaBox, the binding site of EsaR. <br>
+
                <td><strong>Component</strong></td>
−
After the system now has learnt to respond to the associated stimulus alone, the expression of GFP can easily be induced by just exposing it to the stimulus again, e.g. EsaR's ligand.
+
                <td><strong>Concentration</strong></td>
−
</p>
+
                <td><strong>Amount per 1l</strong></td>
−
</div>
+
                </tr>
 +
           
 +
            <tr>
 +
                <td>Bacto-tryptone</td>
 +
                <td>1%</td>
 +
              <td>10g</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>Yeast Extract</td>
 +
                <td>0.5%</td>
 +
                <td>5g</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>NaCl</td>
 +
                <td>1%</td>
 +
                <td>10g</td>
 +
             
 +
            </tr>
 +
    <tr>
 +
                <td>Agar-agar</td>
 +
                <td>1.5%</td>
 +
                <td>15g</td>
 +
             
 +
            </tr>
 +
         
  
−
</div>
 
−
</div>
 
  
−
<div class="sec white">
+
        </tbody>
−
<div>
+
    </table>
−
<h3>Biological Implementation: CRISPR/Cpf1</h3>
+
−
<p>
+
−
An alternative to a recombinase-based switch is the usage of the 2015 characterised <a href="#cpf1" class="cit">CRISPR/Cpf1</a> system. Instead of cutting both DNA strands at the same position, Cpf1 cuts the DNA with an offset of four or five nucleotides, thus producing single-stranded overhangs. It is suggested that this is advantageous for genome editing via non-homologous end-joining.<br>
+
−
We will use the features of Cpf1 to create an AND-gate controlled one-way switch with finally the same functionality as the recombinase-based switch.<br>
+
−
For this, a reporter construct is stably integrated into the genome of <i>E. coli</i> whereas Cpf1 and its guide RNAs will be expressed from a plasmid. <br>
+
−
</p>
+
−
</div>
+
−
</div>
+
  
−
<div class="sec white three_columns">
+
    <p> <br>Sterilized by autoclaving</p>
−
<div>
+
    </div>
  
−
<div>
+
<div class="sec light_grey" id="M9 Salts">
−
<h4>Sensor AND-gate:</h4><br>
+
    <h3>M9 Salts 10x</h3>
−
<p>
+
       
−
At an inflammation spot, nitric oxide activates NorR and triggers the transcription of Cfp1. The transcription can only proceed if 3-OH-C6-HSL is present. The HSL lets the repressor EsaR dissociate from the regulatory element (esaBox) on the DNA and thus annihiliates its roadblock activity. The guide RNAs are expressed constitutively at a high level.<br>
+
−
</p>
+
−
</div>
+
  
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>Concentration</strong></td>
 +
                <td><strong>Amount per 250ml</strong></td>
 +
                </tr>
 +
           
 +
            <tr>
 +
                <td>Na<sub>2</sub>PO<sub>4</sub>&middot; 7 H<sub>2</sub>O</td>
 +
                <td>128g/L</td>
 +
              <td>31.96g</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>KH<sub>2</sub>PO<sub>4</sub></td>
 +
                <td>30 g/L</td>
 +
                <td>7.64g</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>NaCl</td>
 +
                <td>5g/L</td>
 +
                <td>1.248g</td>
 +
             
 +
            </tr>
 +
    <tr>
 +
                <td>NH<sub>4</sub></td>
 +
                <td>10g/L</td>
 +
                <td>2.495g</td>
 +
             
 +
            </tr>
 +
         
  
−
<div>
 
−
<h4>Learning:</h4><br>
 
−
<p>
 
−
Once Cfp1 is expressed, it is brought to the cutting-sites by the two distinct guide RNAs. There, Cfp1 cuts out the <i>mNectarine</i> gene while creating sticky ends. These will then be ligated by endogeneous ligases by NHEJ which reconstitutes the <i>GFP</i> gene.
 
−
<br>
 
−
</p>
 
−
</div>
 
  
 +
        </tbody>
 +
    </table>
  
 +
    <p> <br>Sterilized by autoclaving</p>
 +
    </div>
  
−
<div>
 
−
<h4>Reporter AND-gate:</h4><br>
 
−
<p>
 
−
The reconstituted <i>GFP</i> gene is now under the control of a constitutive promoter regulated be an esaBox, the binding site of EsaR. <br>
 
−
After the system now has learnt to respond to the associated stimulus alone, the expression of GFP can easily be induced by just exposing it to the stimulus again, e.g. EsaR's ligand.
 
−
</p>
 
−
</div>
 
  
−
</div>
+
<div class="sec light_grey" id="LIV solution">
−
</div>
+
    <h3>LIV solution</h3>
 +
       
  
−
<div class="sec dark_grey" id="evolution">
+
        <table>
−
<div>
+
        <tbody>
−
<h2>Directed Evolution of EsaR</h2>
+
            <tr>
−
<p>
+
                <td><strong>Component</strong></td>
−
In order to change EsaR's specificity towards an IBD related HSL, we need to apply directed evolution on this repressor protein.
+
                <td><strong>Concentration</strong></td>
−
The variant of EsaR that we used was already an improved version with a D91G mutation that has an increased signal sensitive compared to the wildtyp<sup><a href="#esar" class="cit">X</a></sup>. We combine several approaches to find new variants of EsaR that are responsive to our target. To select for these variants we have different constructs that form a dual selection system. This systems allows for negative selection ("killing") of variants that still react to the former HSL (3-oxo-C6-HSL) and positive selection ("survival") of variants that respond to the new target HSL (3-OH-C6-HSL). It consists of a fusion protein that is composed of an antibiotic resistance and an enzyme that converts a non-toxic compound into a cellular toxin. We test the combination of the chloramphenicol acetyltransferase (CAT) and the uracil phosphoribosyltransferase (UPRT)<sup><a href="#rackham2005network" class="cit">1</a></sup> as well as the herpes simplex virus thymidine kinase (hsvTK) fused to the aminoglycoside phosphotransferase (APH)<sup><a href="#tominaga2015rapid" class="cit">1</a></sup>. Whereas CAT and APH confer resistance for the positive selection step, UPRT and hsvTK are necessary for the negative selection. <br>
+
                <td><strong>Amount per 10ml</strong></td>
−
UPRT normally converts uracil into uridine monophosphate (dUMP). 5-fluorouracil is metabolised by UPRT to 5-fluoro-dUMP which irreversibly blocks the thymidylate synthase (thyA), a key enzyme for the production of pyrimidine nucleosides in the cell, what finally leads to cell death<sup><a href="#hartmann1961studies" class="cit">1</a></sup>.<br>
+
                </tr>
−
The herpes simplex virus thymidine kinase has a less stringent substrate specificity than normal thymidine kinase and thus also metabolises ganciclovir, a guanin analogue. The metabolised ganciclovir is then treated by the cells as guanin but finally inhibits DNA replication by chain termination.<br>
+
           
−
To generate a library of high diversity, we use site directed mutagenesis as well as random mutagenesis with Taq polymerase and manganese. <br>
+
            <tr>
 +
                <td>L-Leu</td>
 +
                <td>10g/L</td>
 +
              <td>0.1g</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>L-Ile</td>
 +
                <td>3 g/L</td>
 +
                <td>0.03g</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>L-Val</td>
 +
                <td>15g/L</td>
 +
                <td>0.15g</td>
 +
             
 +
            </tr>
  
  
 +
        </tbody>
 +
    </table>
 +
 +
    <p> <br>Sterilized by filtration</p>
 +
    </div>
 +
 +
<div class="sec light_grey" id="Trace elements solution US*">
 +
    <h3>Trace elements solution US*</h3>
 +
       
 +
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>Concentration</strong></td>
 +
                <td><strong>Amount per 50ml</strong></td>
 +
                </tr>
 +
           
 +
            <tr>
 +
                <td>FeSO<sub>4</sub></td>
 +
                <td>5.5g/L</td>
 +
              <td>0.25</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>CaCl<sub>2</sub>&middot; 2 H<sub>2</sub>O</td>
 +
                <td>4.1g/L</td>
 +
                <td>0.2g</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>MnCl<sub>2</sub>&middot; 4 H<sub>2</sub>O</td>
 +
                <td>1.5g/L</td>
 +
                <td>0.075g</td>
 +
             
 +
            </tr>
 +
<tr>
 +
                <td>ZnSO<sub>4</sub></td>
 +
                <td>1.05g/L</td>
 +
                <td>0.0502g</td>
 +
             
 +
            </tr>
 +
<tr>
 +
                <td>H<sub>3</sub>BO<sub>3</sub></td>
 +
                <td>0.3g/L</td>
 +
                <td>0.015g</td>
 +
             
 +
            </tr>
 +
<tr>
 +
                <td>Na<sub>2</sub>[MoSO<sub>4</sub>]&middot; 2 H<sub>2</sub>O</td>
 +
                <td>0.25g/L</td>
 +
                <td>0.013g</td>
 +
             
 +
            </tr>
 +
 +
<tr>
 +
                <td>CuCl<sub>2</sub>&middot; 2 H<sub>2</sub>O</td>
 +
                <td>0.15g/L</td>
 +
                <td>0.0075g</td>
 +
             
 +
            </tr>
 +
<tr>
 +
                <td>Na<sub>2</sub>[EDTA]&middot; 2 H<sub>2</sub>O</td>
 +
                <td>0.84g/L</td>
 +
                <td>0.042g</td>
 +
             
 +
            </tr>
 +
 +
        </tbody>
 +
    </table>
 +
 +
    <p> <br>Sterilized by filtration</p>
 +
    </div>
 +
 +
<div class="sec light_grey" id="M9* LIV Medium">
 +
    <h3>M9* LIV Medium</h3>
 +
       
 +
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>Concentration</strong></td>
 +
                <td><strong>Volume for 1L</strong></td>
 +
                </tr>
 +
           
 +
            <tr>
 +
                <td>M9 Salts</td>
 +
                <td>10x</td>
 +
              <td>100ml</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>Glucose</td>
 +
                <td>50%</td>
 +
                <td>20ml</td>
 +
             
 +
            </tr>
 +
            <tr>
 +
                <td>MgSO<sub>4</sub></td>
 +
                <td>1M</td>
 +
                <td>2ml</td>
 +
             
 +
            </tr>
 +
<tr>
 +
                <td>CaCl<sub>2</sub></td>
 +
                <td>0.1M</td>
 +
                <td>1ml</td>
 +
             
 +
            </tr>
 +
<tr>
 +
                <td>Antibiotic</td>
 +
                <td>1000x</td>
 +
                <td>1ml</td>
 +
             
 +
            </tr>
 +
<tr>
 +
                <td>Trace elements solution US*</td>
 +
                <td>1000x</td>
 +
                <td>1ml</td>
 +
             
 +
            </tr>
 +
 +
<tr>
 +
                <td>Thiamine</td>
 +
                <td>10g/L</td>
 +
                <td>2ml</td>
 +
             
 +
            </tr>
 +
<tr>
 +
                <td>LIV Solution</td>
 +
                <td>100x</td>
 +
                <td>10ml</td>
 +
             
 +
            </tr>
 +
<tr>
 +
                <td>H<sub>2</sub>O</td>
 +
                <td>55.5M</td>
 +
                <td>863ml</td>
 +
             
 +
            </tr>
 +
 +
        </tbody>
 +
    </table>
 +
 +
    </div>
 +
 +
<div class="sec light_grey" id="HEPES Buffer">
 +
    <h3>HEPES Buffer</h3>
 +
       
 +
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>Concentration</strong></td>
 +
                <td><strong>Amount per 50ml</strong></td>
 +
                </tr>
 +
           
 +
            <tr>
 +
                <td>NaOH</td>
 +
                <td>5M</td>
 +
              <td>10g</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>HEPES</td>
 +
                <td>100mM</td>
 +
                <td>0.475g</td>
 +
             
 +
            </tr>
 +
           
 +
         
 +
 +
 +
        </tbody>
 +
    </table>
 +
 +
    <p> <br>Adjust pH to 7.0 and sterilize by filtration</p>
 +
    </div>
 +
 +
 +
<div class="sec light_grey" id="ITA Buffer">
 +
    <h3>Isothermal Reaction Buffer </h3>
 +
       
 +
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>Concentration</strong></td>
 +
                <td><strong>Amount per 15ml</strong></td>
 +
                </tr>
 +
           
 +
            <tr>
 +
                <td>Tris-HCl pH7.5</td>
 +
                <td>1M</td>
 +
              <td>3ml</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>MgCl<sub>2</sub></td>
 +
                <td>2M</td>
 +
                <td>150ul</td>
 +
             
 +
            </tr>
 +
           
 +
          <tr>
 +
                <td>dNTP</td>
 +
                <td>100mM</td>
 +
                <td>60ul</td>
 +
             
 +
            </tr>
 +
 +
          <tr>
 +
                <td>DTT</td>
 +
                <td>1M</td>
 +
                <td>300ul</td>
 +
             
 +
            </tr>
 +
 +
          <tr>
 +
                <td>NAD</td>
 +
                <td>100mM</td>
 +
                <td>300ul</td>
 +
             
 +
            </tr>
 +
 +
          <tr>
 +
                <td>PEG</td>
 +
                <td>N/A</td>
 +
                <td>1.5g</td>
 +
             
 +
            </tr>
 +
 +
 +
        </tbody>
 +
    </table>
 +
 +
    <p> <br>Sterilized by filtration</p>
 +
    </div>
 +
 +
<div class="sec light_grey" id="Gibson Assembly mix">
 +
    <h3>Gibson Assembly Mix</h3>
 +
       
 +
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>Concentration</strong></td>
 +
                <td><strong>Amount per 15ml</strong></td>
 +
                </tr>
 +
           
 +
            <tr>
 +
                <td>ITA Buffer</td>
 +
                <td>3.5x</td>
 +
              <td>350ul</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>H<sub>2</sub>O</td>
 +
                <td>55.5M</td>
 +
                <td>700ul</td>
 +
             
 +
            </tr>
 +
           
 +
          <tr>
 +
                <td>T5 Exonuclease</td>
 +
                <td>10,000units/ml</td>
 +
                <td>0.64ul</td>
 +
             
 +
            </tr>
 +
 +
          <tr>
 +
                <td>Phusion DNA Pol</td>
 +
                <td>2,000units/ml</td>
 +
                <td>20ul</td>
 +
             
 +
            </tr>
 +
 +
          <tr>
 +
                <td>Taq DNA Ligase</td>
 +
                <td>40,000units/ml</td>
 +
                <td>160ul</td>
 +
             
 +
            </tr>
 +
 +
 +
        </tbody>
 +
    </table>
 +
 +
    <p> <br>Transferred into PCR tubes in 15ul aliquots</p>
 +
    </div>
 +
 +
<div class="sec light_grey" id="DETA/NO Solution">
 +
    <h3>DETA/NO 650mM Solution</h3>
 +
       
 +
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>Concentration</strong></td>
 +
                <td><strong>Amount per 1ml</strong></td>
 +
                </tr>
 +
           
 +
            <tr>
 +
                <td>DETA/NO</td>
 +
                <td>N/A</td>
 +
              <td>10mg</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>NaOH</td>
 +
                <td>0.1M</td>
 +
                <td>Up to 1ml</td>
 +
             
 +
            </tr>
 +
           
 +
 +
 +
        </tbody>
 +
    </table>
  
−
</p>
 
−
</div>
 
 
</div>
 
</div>
 +
<div class="sec light_grey" id="AHL Solution">
 +
    <h3>AHL Solution 100mM</h3>
 +
       
  
 +
        <table>
 +
        <tbody>
 +
            <tr>
 +
                <td><strong>Component</strong></td>
 +
                <td><strong>Concentration</strong></td>
 +
                <td><strong>Amount per 1ml</strong></td>
 +
                </tr>
 +
           
 +
            <tr>
 +
                <td>N-Hexanoyl-L-Homoserine Lactone</td>
 +
                <td>N/A</td>
 +
              <td>19.9mg</td>
 +
            </tr>
 +
         
 +
            <tr>
 +
                <td>DMSO</td>
 +
                <td>N/A</td>
 +
                <td>Up to 1ml</td>
 +
             
 +
            </tr>
 +
           
  
−
<div class="sec dark_grey">
+
 
−
<div>
+
        </tbody>
−
<h3>Dual Selection Procedure</h3>
+
    </table>
−
</div>
+
 
 +
    </div>
 
</div>
 
</div>
  
−
<div class="sec dark_grey two_columns">
+
<div class="sec white" id="Measurements">
 
<div>
 
<div>
−
<div>
+
<h2>Measurements</h2>
−
<h4>Negative Selection:</h4><br>
+
<h3>Plate reader experiment</h3>
−
<div class="image_box" style="max-width: 400px;">
+
−
    <a href="https://static.igem.org/mediawiki/2016/8/89/T--ETH_Zurich--NegativeSelection.svg">
+
−
        <img src="https://static.igem.org/mediawiki/2016/8/89/T--ETH_Zurich--NegativeSelection.svg">
+
−
    </a>
+
−
</div>
+
 
<p>
 
<p>
−
 
+
<ul>
−
In a first step, the created library of variants is grown in presence of the old inducer 3-oxo-C6-HSL and a toxic precursor.
+
<li>
−
Variants whose expression is still induced by the old HSL or have a non-functional repressor (EsaR) express the fusion protein which converts the toxic precursor into a toxin (A), non-responsive repressors stay bound to the DNA and inhibit protein expression (B). <br>
+
<h4>Day -1: Restreak</h4>
−
After a certain time, the surviving variants are transfered into culture medium without the toxic precurser and without HSL in order to eliminate the fusion protein.
+
<p>
 +
Restreak all experiments you want to run from cryostocks
 
</p>
 
</p>
−
</div>
+
</li>
−
<div>
+
<li><h4>Day 0: Prepare precultures</h4>
−
<h4>Positive Selection:</h4><br>
+
−
<div class="image_box" style="max-width: 400px;">
+
−
    <a href="https://static.igem.org/mediawiki/2016/8/87/T--ETH_Zurich--PositiveSelection.svg">
+
−
        <img src="https://static.igem.org/mediawiki/2016/8/87/T--ETH_Zurich--PositiveSelection.svg">
+
−
    </a>
+
−
</div>
+
 
<p>
 
<p>
 +
Prepare precultures of all the experiments you want to run. Prepare 5mL of freshly prepared M9 medium (prepared the same morning), necessary antibiotics, and pick one colony from the plate. Ideally you want to run three biological replicates, but this can be done in three different experiments (the 96-well plate has a finite number of wells and your experiment might not fit). Priority is technical replicates (see setting up the experiment). Keep M9 the in +4 frigde until next day.
 +
</p>
 +
</li>
 +
<li><h4>Day 1</h4>
 +
<p>
 +
<ul>
 +
<li>Prepare and divide into PCR tubes all PBS amounts necessary to make the dilutions of DETA/NO from the stock. Place PCR tubes in a row next to each other. This eases work with multichannel pipette. Use “experimental calculations” excel sheet to calculate DETA/NO dilutions.
 +
</li>
 +
<li> Open Tecan iController. Open "Heating" on settings and click ON to set temperature to37 degrees before the experiment starts.
 +
</li>
 +
<li><h5>OD600 measurement and dilution</h5>
 +
<p>
 +
<ul>
 +
<li>Measure OD600 using the spectrometer. Place 1 mL of liquid (overnight culture) in the cuvette, make sure the arrow on the cuvette aligns with the arrow on the machine. Also don’t touch the lower parts of the cuvette as it can interfere with the measurement.
 +
</li>
 +
<li> Blank spectrometer with M9. If it is not possible to measure OD, dilute the culture 1:1 and try again.
 +
</li>
 +
<li>Dilute preculture to OD=0.1 with M9 and add antibiotics. Take a single packaged sterile 96-well plate. Pipette 200 uL per well and seal completely without touching the seal. Place the plate in the plate reader. Keep in mind the plate reader OD is 1/4th of the actual OD.
 +
</li>
 +
<li>Dilute precultures and let them shake in the plate reader for at least 2 hours before induction to make sure cells are in the exponential phase during induction. Add a blank with M9 media in the well plate.
 +
</li>
 +
</ul>
 +
</li>
 +
<li><h5>Setting up the experiment</h5>
 +
<h5>Design</h5>
 +
<p>
 +
<ul>
 +
<li>
 +
Design experiment such that you have triplicates of each sample to be tested.
 +
</li>
 +
<li>
 +
On Tecan iController, select the type of plate you are using. The standard one is Greiner 96 Flat Transparent.
 +
</li>
 +
<li>Set the parameters of the Tecan plate reader protocol:
 +
<ul>
 +
<li>Temperature: 37 degrees. </li>
 +
<li>Shaking: 10 seconds, Amplitude 6, Mode: orbital. </li>
 +
<li> Kinetic Cycle: pick 100 cycles</li>
 +
<li>Set OD measurement to 600. Set number of flashes to 25. </li>
 +
<li>add shaking step for 10 seconds with amplitude 6 </li>
 +
<li> add Fluorescence Intensity. For GFP set excitation to 488nm and emission toemission 530nm.  The numbers that appear next to nm is the +/- range of the excitation/emission. So pay attention that the range of excitation and emission you get never overlaps, or the measurements will not be accurate. Number of flashes:25. Settle time:0ms. Mode: Top. Z-position: Manual, 20000um. Lag time 0us. Integration time 20us. Label: Green Fluorescence. </li>
 +
<li>add additional shaking step of 900 seconds with amplitude 6mm and orbital mode and hit start. </li>
 +
</ul>
 +
As soon as you out the plate in the plate reader, prepare serial dilutions of DETA/no in PBS.
 +
</li>
 +
</ul>
 +
</p>
 +
</ul>
 +
</li>
 +
<li><h5>Measurements: </h5>
 +
<ul>
 +
<li>Measurements are collected every 15 minutes. They will be recorded in an excel sheet that automatically opens. It is not recommended to work on tahat excel sheet while the experiment is running. </li>
 +
<li> Observe OD measurements over time. Always substract it from the M9 blank wells to decide whether or not you have reached the desired OD. You should induce at real OD=0.5 (plate reader OD=0.125). After inducing, put on a new seal. Otherwise condensation interferes with the measurement and also you might cross-contaminate.
 +
</li>
 +
</ul>
 +
</p>
  
−
In a second step, these variants now undergo a round of positive selection to select for variants that are responsive to the new HSL 3-OH-C6-HSL. <br>
+
<h3>Fluorescence-activated cell sorting (FACS)</h3>
−
The bacteria are cultured in medium containing the new HSL and the antibiotic whose resistance is part of the fusionprotein. Inducible variants express the resistance protein and survive (A). Variants that are non-responsive do not express it and can not grow (B). <br>
+
<p>
−
Afterwards, the variants can be plated and analyzed or undergo further rounds of positive / negative selection to enrich for suitable variants.
+
The protocol was designed by Lukas with the help and consultation of advisers.  
 +
<ul>
 +
<li>Inoculate 5 mL of overnight culture</li>
 +
<li>Dilute cells to OD 0.1 in 50 mL flask</li>
 +
<li>Let cells grow to OD 0.5 and split them into 6 inoculation tubes, with 6 mL of culture per tube</li>
 +
<li>Induce tubes with appropriate concentrations</li>
 +
<li>Take samples at different time points:
 +
<ul>
 +
<li>Take sample to measure OD and note it</li>
 +
<li>Take another sample (100 uL): centrifuge it down (4 degrees, 10 000 g, 2 min)</li>
 +
<li>Resuspend sample in 1 mL 1x PBS</li>
 +
<li>Dilute sample to OD 0.01 and store it on ice</li>
 +
</ul>
 +
</li>
 +
<li>Transfer samples on 96-well plates</li>
 +
</ul>
 +
Note: A separate tube (1 mL) with negative control (no fluorescence) and positive control with very strong fluorescence for calibration (OD = 0.005) also has to be prepared.
 
</p>
 
</p>
−
</div>
+
 
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<div class="sec blue">
+
<div class="sec light_grey" id="Kits">
 
<div>
 
<div>
−
<h4>Potential Delivery Method of Reporter Strains</h4>
+
<h2>Kits</h2>
−
 
+
<h3>Gel recovery</h3>
−
The idea to administer genetically modified bacteria in the context of IBD was published by Steidler <i>et al.</i> in 2000<sup><a href="#steidler2000treatment" class="cit">1</a></sup>. Generally, to use engineered probiotic bacteria as a delivery vector for <i>in vivo</i> produced therapeutic agents has been described multiple times<sup><a href="#bermudez2009lactococcus" class="cit">1</a>,<a href="#bermudez2013engineering" class="cit">1</a>,<a href="#wells2008mucosal" class="cit">1</a>,<a href="#deming2013genetically" class="cit">1</a></sup>. <br>
+
<h4>Zymoclean Gel DNA Recovery Kit</h4>
−
As the stomach and the gastrointestinal tract are rough environments for non-adapted (probiotic) bacteria, we suggest to encapsulate the bacteria in a hydrogel. This protects the bacteria and ensures the recovery of the reporter strain. The method of encapsulation is well known for oral administration in animal models (e.g. Prakash <i>et al.</i><sup><a href="#prakash1996microencapsulated" class="cit">1</a></sup>) and is summarised in several reviews (e.g. Prakash <i>et al.</i> (2008)</i><sup><a href="#prakash2008colon" class="cit">1</a></sup> and Tomaro <i>et al.</i> (2012) <sup><a href="#tomaro2012microencapsulation" class="cit">1</a></sup>).
+
<p>
 +
The kit is used to recover DNA fragments from agarose gel.
 +
<ul>
 +
<li>Supplier: Zymo Research</li>
 +
<li>Catalog No: D4002</li>
 +
</ul>
 +
Instead of the elution buffer provided in the kit we use Tris-HCl solution heated to 65C to elute DNA.
 +
</p>
  
 +
<h3>Plasmid extraction and purification</h3>
 +
<h4>ZR Plasmid MiniprepTM - Classic</h4>
 +
<p>
 +
The kit is used to isolate plasmid from bacteria.
 +
<ul>
 +
<li>Supplier: Zymo Research</li>
 +
<li>Catalog No: D4016</li>
 +
</ul>
 +
Instead of the elution buffer provided in the kit we use Tris-HCl solution heated to 65C to elute DNA.
 +
</p>
 
</div>
 
</div>
 
</div>
 
</div>
−
 
−
 
−
 
  
  
Line 827: Line 1,415:
  
  
−
<li><a name="scaldaferri2015gut" class></a>[1] Scaldaferri, F., et al. "Gut microbiota molecular spectrum in healthy controls, diverticular disease, IBS and IBD patients: Time for microbial marker of gastrointestinal disorders?." Journal of Crohns & Colitis. Vol. 9. Oxford Univ Press, 2015.
+
<li><a name="gibson" class></a>[2] Gibson, Daniel G., et al. "Enzymatic assembly of DNA molecules up to several hundred kilobases." Nature methods 6.5 (2009): 343-345.
 
 
 
   
 
   
Line 839: Line 1,427:
  
  
−
 
+
</div>
−
 
+
</div>
−
 
+
  
 
</body>
 
</body>
 
</html>
 
</html>
 +
 
{{:Template:ETH_Zurich/footer}}
 
{{:Template:ETH_Zurich/footer}}

Latest revision as of 20:40, 19 October 2016

PAVLOV'S COLI

Experimental: Protocols, Methods and Material

Polymerase Chain Reaction - for Construction

New England Biolabs Phusion High-Fidelity DNA Polymerase

Composition:

Component 50 µl Reaction
Phusion HF Buffer (5x) 10 µl
dNTPs (10 mM) 1 µl
Forward Primer (10 µM) 2.5 µl
Reverse Primer (10 µM) 2.5 µl
Template DNA variable
DMSO (optional) up to 3%
Nuclease-free water up to 50 µl
Phusion Polymerase 0.5 µl

Thermocycling Conditions:

Temperature
Time
98°C 30 seconds
98°C
45-72°C
72°C 		5-10 seconds
10-30 seconds
15-30 seconds per kb
72°C 5-10 minutes


The appropriate annealing temperature was calculated from NEB's Tm Calculator

Kapa Biosystems Hifi Hotstart Ready Mix

Component 50 µl Reaction
Master Mix (2x) 25 µl
Forward Primer (10 µM) 2.5 µl
Reverse Primer (10 µM) 2.5 µl
Template DNA variable
Nuclease-free water up to 50 µl

Thermocycling Conditions:

Temperature
Time
95°C 3 minutes
98°C
55-75°C
72°C 		20 seconds
15 seconds
15-60 seconds per kb
72°C 1 minute per kb


Kapa Hifi Hotstart has similar annealing temperatures as Phusion DNA polymerase, even slightly higher. Only in very few cases a lower annealing temperature was found to be better.

Polymerase Chain Reaction - Colony PCR

For screening a large number of clones, single colonies were resuspended in 20 µl LB medium of which 1 µl was used as template for the PCR.

Solis BioDyne FirePol DNA Polymerase

Composition:

Component 8 x 20 µl Reaction (+ 10 µl excess)
FIREPol DNA Polymerase 0.85 µl
MgCl2 (25 mM) 10.2 µl
Reaction Buffer B (10x) 17 µl
dNTPs (10 mM) 3.4 µl
Forward Primer (10 µM) 3.4 µl
Reverse Primer (10 µM) 3.4 µl
Template DNA 1 µl per 20 µl reaction
Nuclease-free water 123.25 µl

Thermocycling Conditions:

Temperature
Time
98°C 3-5 minutes
95°C
50-72°C
72°C 		30-60 seconds
30-60 seconds
1 minute per kb
72°C 5-10 minutes

New England Biolabs Taq DNA Polymerase

Composition:

Component 8 x 20 µl Reaction (+ 10 µl excess)
Taq Reaction Buffer (10x) 17 µl
dNTPs (10 mM) 3.4 µl
Forward Primer (10 µM) 3.4 µl
Reverse Primer (10 µM) 3.4 µl
Template DNA 1 µl per 20 µl reaction
Taq DNA Polymerase 0.85 µl
Nuclease-free water 133.45 µl

Thermocycling Conditions:

Temperature
Time
95°C 5 minutes
95°C
45-68°C
68°C 		30 seconds
20 seconds
1 minute per kb
72°C 5 minutes


The appropriate annealing temperature was calculated from NEB's Tm Calculator

Site-Directed Mutagenesis (QuickChange)

Component 50 µl Reaction
Kapa Hifi Hotstart Master Mix (2x) 25 µl
Forward Primer (10 µM) 2.5 µl
Reverse Primer (10 µM) 2.5 µl
Template DNA variable
Nuclease-free water up to 50 µl

Thermocycling Conditions:

Temperature
Time
95°C 3 minutes
98°C
65°C
72°C 		20 seconds
15 seconds
15-60 seconds per kb
72°C 1 minute per kb


Primers were designed according to the guidlines of The Richard Lab 1

  • The targeted mutation should be included into both primers.
  • The mutation can be as close as 4 bases from the 5'-terminus.
  • The mutation should be at least 8 bases from the 3'-terminus.
  • At least eight non-overlapping bases should be introduced at the 3-end of each primer.
  • At least one G or C should be at the end of each primer.
  • Design your primers (including the mutations) to have a Tm >=78°C.

The resulting PCR product has to be purified (e.x. Agencourt AMPure XP) and digested with DpnI (NEB) for 4 hours and gel-purified. In case Phusion polymerase is used, DpnI can directly be added to the PCR. The product can then immediately be used for transformation. In order to obtain high base exchange efficiency, the template need to be removed from the reaction thoroughly.

Isothermal "Gibson" Assembly2

Recipe for Ready-to-Use Isothermal Assembly Mixes

5x Isothermal Reaction Buffer (6ml)

  • 3 ml of 1 M Tris-HCl pH 7.5
  • 150 µl of 2 M MgCl2
  • 60 µl of 100 mM dGTP
  • 60 µl of 100 mM dATP
  • 60 µl of 100 mM dTTP
  • 60 µl of 100 mM dCTP
  • 300 µl of 1 M DTT
  • 1.5 g PEG-8000
  • 300 µl of 100 mM NAD


This buffer can be aliquoted and stored at -20 °C.

Isothermal Assembly Master Mix

  • 320 µl 5x isothermal reaction buffer
  • 0.64 µl of 10 U/µl T5 exonuclease
  • 20 µl of 2 U/µl Phusion DNA polymerase
  • 160 µl of 40 U/µl Taq DNA ligase
  • Fill up with water to a final volume of 1.2 ml


The master mix is devided into aliquots of 15 µl and stored at -20 °C.

Protocol for Isothermal Assembly

  • 15 µl aliquot of master mix
  • 0.02-0.5 pmol DNA in total for 2-3 fragments
      or
  • 0.2-1 pmol DNA in total for 4-6 fragments
  • Fill up with water to 20 µl
Consider the following:
  • 2-3 times more insert than backbone (molar ratio)
  • 5 times more insert for fragments < 200 bp (molar ratio)


The assembled mix is then incubated for 60 minutes at 50 °C. It can be directly transformed with chemically competent cell (volume of assembly reaction not exceeding 10% of the volume of the competent cells). Otherwise, the reaction mix is purified and desalted (e.x. Agencourt AMPure XP) and a fraction of it transformed with electrocompetent cells.

Preparation of Competent Cells

Preparation of Chemically Competent Cells

  • Inoculate 100 ml of prewarmed LB medium with 1 ml overnight culture and grow the bacteria to an OD600 of 0.5.
  • Cool the culture on ice, transfer the cells into cetrifugation tubes and harvest them by centrifugation for 5 min (4000xg, 4°C)
  • Carfully discard supernatant, keep cells always on ice.
  • Resuspend cells in 30 ml cold TFB1 and incubate on ice for 90 minutes.
  • Centrifuge for 5 min (4000xg, 4°C)
  • Carfully discard supernatant, keep cells always on ice.
  • Resuspend the cells in 4 ml ice-cold TFB2 buffer.
  • Prepare aliquots of 100 µl in pre-cooled, sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at -80° C

Transformation Protocol for Chemically Competent Cells

  • Thaw the competent cells on ice
  • Use 50 ul of the competent cells for one transformation, do not add more than 5 ul of DNA to the cells (10% of the volume of the competent cells)
  • Incubate them for 20 min on ice
  • Incubate them for 90 s at 42° C and add afterwards 500 µl of SOC
  • Let them regenerate for 1 h at 37° C with shaking
  • Plate a part of the culture according to your expectations on LB-agar plates with the approbriate antibiotic.

Preparation of Electrocompetent Cells

  • Inoculate 600 ml of prewarmed LB medium with 6 ml overnight culture and grow the bacteria to an OD600 of 0.4.
  • Cool the culture on ice fpr 30 min, transfer 300 ml of the culture into 50 ml tubes and centrifuge them for 10 min (3000xg, 4°C). Discard supernatant and add the rest of the culture. Again discard supernatant.
  • Carfully resuspend cells in ice-cold, autoclaved and deionized water and fill up to 50 ml
  • Centrifuge again for 10 min (3000xg, 4°C) and repeat wash step from above
  • Carfully resuspend cells in ice-cold 10% glycerol (working always on ice)
  • Centrifuge for 10 min (3000xg, 4°C)
  • Discard supernatant
  • Resuspend the cells in the remaining glycerol solution.
  • Prepare aliquots of 35 µl in pre-cooled, sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at -80° C

Transformation Protocol for Electrocompetent Cells

  • Thaw the electrocompetent cells on ice. Pre-chill also the electroporation cuvette.
  • Add DNA to the cells. Attention: the DNA should contain as little ions as possible. Purify or dilute reactions containing salt.
  • Transfer the bacteria/DNA mix into a pre-chilled electroporation cuvette and electroporate them.
  • Add IMMEDIATELY 500 µl of SOC and transfer them back into the microcentrifuge tube.
  • Let them regenerate for 1 h at 37° C with sufficient shaking.
  • Plate a part of the transformation mix according to your expectations on LB-agar plates with the approbriate antibiotic.

Buffer and Medium

TFB1

Component Concentration Amount per 100 ml
RbCl 100 mM 1.21 g
MnCl2 · 4 H2O 50 mM 0.99 g
Potassium acetate · 4 H2O 30 mM 0.29 g
CaCl2 · 2 H2O 10 mM 0.147 g
Glycerol 15 % 11.9 g


Adjust pH to 5.5 and sterilize by filtration

TFB2

Component Concentration Amount per 100 ml
MOPS 100 mM 0.0047 g
RbCl 50 mM 0.0027 g
CaCl2 · 2 H2O 75 mM 0.024 g
Glycerol 15 % 0.27g


Adjust pH to 6.5 and sterilize by filtration

LB Medium

Component Concentration Amount per 1l
Bacto-tryptone 1% 10g
Yeast Extract 0.5% 5g
NaCl 1% 10g


Sterilized by autoclaving

LB Agar

Component Concentration Amount per 1l
Bacto-tryptone 1% 10g
Yeast Extract 0.5% 5g
NaCl 1% 10g
Agar-agar 1.5% 15g


Sterilized by autoclaving

M9 Salts 10x

Component Concentration Amount per 250ml
Na2PO4· 7 H2O 128g/L 31.96g
KH2PO4 30 g/L 7.64g
NaCl 5g/L 1.248g
NH4 10g/L 2.495g


Sterilized by autoclaving

LIV solution

Component Concentration Amount per 10ml
L-Leu 10g/L 0.1g
L-Ile 3 g/L 0.03g
L-Val 15g/L 0.15g


Sterilized by filtration

Trace elements solution US*

Component Concentration Amount per 50ml
FeSO4 5.5g/L 0.25
CaCl2· 2 H2O 4.1g/L 0.2g
MnCl2· 4 H2O 1.5g/L 0.075g
ZnSO4 1.05g/L 0.0502g
H3BO3 0.3g/L 0.015g
Na2[MoSO4]· 2 H2O 0.25g/L 0.013g
CuCl2· 2 H2O 0.15g/L 0.0075g
Na2[EDTA]· 2 H2O 0.84g/L 0.042g


Sterilized by filtration

M9* LIV Medium

Component Concentration Volume for 1L
M9 Salts 10x 100ml
Glucose 50% 20ml
MgSO4 1M 2ml
CaCl2 0.1M 1ml
Antibiotic 1000x 1ml
Trace elements solution US* 1000x 1ml
Thiamine 10g/L 2ml
LIV Solution 100x 10ml
H2O 55.5M 863ml

HEPES Buffer

Component Concentration Amount per 50ml
NaOH 5M 10g
HEPES 100mM 0.475g


Adjust pH to 7.0 and sterilize by filtration

Isothermal Reaction Buffer

Component Concentration Amount per 15ml
Tris-HCl pH7.5 1M 3ml
MgCl2 2M 150ul
dNTP 100mM 60ul
DTT 1M 300ul
NAD 100mM 300ul
PEG N/A 1.5g


Sterilized by filtration

Gibson Assembly Mix

Component Concentration Amount per 15ml
ITA Buffer 3.5x 350ul
H2O 55.5M 700ul
T5 Exonuclease 10,000units/ml 0.64ul
Phusion DNA Pol 2,000units/ml 20ul
Taq DNA Ligase 40,000units/ml 160ul


Transferred into PCR tubes in 15ul aliquots

DETA/NO 650mM Solution

Component Concentration Amount per 1ml
DETA/NO N/A 10mg
NaOH 0.1M Up to 1ml

AHL Solution 100mM

Component Concentration Amount per 1ml
N-Hexanoyl-L-Homoserine Lactone N/A 19.9mg
DMSO N/A Up to 1ml

Measurements

Plate reader experiment

  • Day -1: Restreak

    Restreak all experiments you want to run from cryostocks

  • Day 0: Prepare precultures

    Prepare precultures of all the experiments you want to run. Prepare 5mL of freshly prepared M9 medium (prepared the same morning), necessary antibiotics, and pick one colony from the plate. Ideally you want to run three biological replicates, but this can be done in three different experiments (the 96-well plate has a finite number of wells and your experiment might not fit). Priority is technical replicates (see setting up the experiment). Keep M9 the in +4 frigde until next day.

  • Day 1

    • Prepare and divide into PCR tubes all PBS amounts necessary to make the dilutions of DETA/NO from the stock. Place PCR tubes in a row next to each other. This eases work with multichannel pipette. Use “experimental calculations” excel sheet to calculate DETA/NO dilutions.
    • Open Tecan iController. Open "Heating" on settings and click ON to set temperature to37 degrees before the experiment starts.
    • OD600 measurement and dilution

      • Measure OD600 using the spectrometer. Place 1 mL of liquid (overnight culture) in the cuvette, make sure the arrow on the cuvette aligns with the arrow on the machine. Also don’t touch the lower parts of the cuvette as it can interfere with the measurement.
      • Blank spectrometer with M9. If it is not possible to measure OD, dilute the culture 1:1 and try again.
      • Dilute preculture to OD=0.1 with M9 and add antibiotics. Take a single packaged sterile 96-well plate. Pipette 200 uL per well and seal completely without touching the seal. Place the plate in the plate reader. Keep in mind the plate reader OD is 1/4th of the actual OD.
      • Dilute precultures and let them shake in the plate reader for at least 2 hours before induction to make sure cells are in the exponential phase during induction. Add a blank with M9 media in the well plate.
    • Setting up the experiment
      Design

      • Design experiment such that you have triplicates of each sample to be tested.
      • On Tecan iController, select the type of plate you are using. The standard one is Greiner 96 Flat Transparent.
      • Set the parameters of the Tecan plate reader protocol:
        • Temperature: 37 degrees.
        • Shaking: 10 seconds, Amplitude 6, Mode: orbital.
        • Kinetic Cycle: pick 100 cycles
        • Set OD measurement to 600. Set number of flashes to 25.
        • add shaking step for 10 seconds with amplitude 6
        • add Fluorescence Intensity. For GFP set excitation to 488nm and emission toemission 530nm. The numbers that appear next to nm is the +/- range of the excitation/emission. So pay attention that the range of excitation and emission you get never overlaps, or the measurements will not be accurate. Number of flashes:25. Settle time:0ms. Mode: Top. Z-position: Manual, 20000um. Lag time 0us. Integration time 20us. Label: Green Fluorescence.
        • add additional shaking step of 900 seconds with amplitude 6mm and orbital mode and hit start.
        As soon as you out the plate in the plate reader, prepare serial dilutions of DETA/no in PBS.

  • Measurements:
    • Measurements are collected every 15 minutes. They will be recorded in an excel sheet that automatically opens. It is not recommended to work on tahat excel sheet while the experiment is running.
    • Observe OD measurements over time. Always substract it from the M9 blank wells to decide whether or not you have reached the desired OD. You should induce at real OD=0.5 (plate reader OD=0.125). After inducing, put on a new seal. Otherwise condensation interferes with the measurement and also you might cross-contaminate.

    Fluorescence-activated cell sorting (FACS)

    The protocol was designed by Lukas with the help and consultation of advisers.

    • Inoculate 5 mL of overnight culture
    • Dilute cells to OD 0.1 in 50 mL flask
    • Let cells grow to OD 0.5 and split them into 6 inoculation tubes, with 6 mL of culture per tube
    • Induce tubes with appropriate concentrations
    • Take samples at different time points:
      • Take sample to measure OD and note it
      • Take another sample (100 uL): centrifuge it down (4 degrees, 10 000 g, 2 min)
      • Resuspend sample in 1 mL 1x PBS
      • Dilute sample to OD 0.01 and store it on ice
    • Transfer samples on 96-well plates
    Note: A separate tube (1 mL) with negative control (no fluorescence) and positive control with very strong fluorescence for calibration (OD = 0.005) also has to be prepared.

Kits

Gel recovery

Zymoclean Gel DNA Recovery Kit

The kit is used to recover DNA fragments from agarose gel.

  • Supplier: Zymo Research
  • Catalog No: D4002
Instead of the elution buffer provided in the kit we use Tris-HCl solution heated to 65C to elute DNA.

Plasmid extraction and purification

ZR Plasmid MiniprepTM - Classic

The kit is used to isolate plasmid from bacteria.

  • Supplier: Zymo Research
  • Catalog No: D4016
Instead of the elution buffer provided in the kit we use Tris-HCl solution heated to 65C to elute DNA.

References:

  • [1] Tom Richard, Department of Agricultural and Biological Engineering, Penn State University.
  • [2] Gibson, Daniel G., et al. "Enzymatic assembly of DNA molecules up to several hundred kilobases." Nature methods 6.5 (2009): 343-345.

Thanks to the sponsors that supported our project: