Difference between revisions of "Team:Munich/Demonstrate"

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<tr><td colspan=6 align=center valign=center>
 
<tr><td colspan=6 align=center valign=center>
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<h3></h3>
+
<h3>Sample processing</h3>
 
<p>
 
<p>
−
We chose to combine heat lysis and isothermal amplification (RPA) to extract our target RNA from patient samples
+
We chose to combine heat lysis and isothermal amplification (RPA) to extract our target RNA from patient samples.
 
</p>
 
</p>
 
   <ul class="listResults">
 
   <ul class="listResults">
−
           <li><a href="/Team:Munich/Cas13a">Demonstrated functionality of Lbu and Lwa Cas13a.</a></li>
+
           <li>We used RPA to amplify DNA from heat lysed <i>E. coli</i>.</li>
−
          <li><a href="/Team:Munich/Cas13a">Modelled the detection limit of our circuit and confirmed it experimentally (~10 nM RNA).</a></li>
+
           <li>We conducted RPA and transcription from an in vitro DNA on paper.</li>
−
          <li><a href="/Team:Munich/Cas13a">Detected pathogen RNA sequence from <i> in vitro </i> and <i> in vivo </i> sources.</a></li>
+
           <li>We amplified and transcribed an in vitro DNA target to RNA concentrations detectable by our readout circuit
−
           <li><a href="/Team:Munich/Targets">Differentiated viral sequences from bacterial sequences.</a></li>
+
.</li>
−
          <li><a href="/Team:Munich/Readouts">Used RNase Alert and Spinach aptamer read-out circuits.</a></li>
+
−
          <li><a href="/Team:Munich/Readouts">Used gold nanoparticles to detect general RNase activity.</a></li>
+
−
          <li><a href="/Team:Munich/Detection">Constructed a functional fluorescence detector with high sensitivity and low production cost.</a></li>
+
−
          <li><a href="/Team:Munich/Cas13a">Detected RNA in bulk, on paper, and from lyophilized Cas13a.</a></li>
+
−
           <li><a href="/Team:Munich/Amplification">Amplified target with RPA and transcription on paper.</a></li>
+
−
          <li><a href="/Team:Munich/Parts">Improved the biobrick BBa_K1319008 by adding a 6x His-tag and provided Cas13a Lwa as three different composite biobricks.</a></li>
+
−
          <li><a href="/Team:Munich/Part_Collection">Characterized the GFP degradation tags and sent them as a part collection.</a></li>
+
 
   </ul>
 
   </ul>
 +
<p>
 +
We validated the three necessary modules of the sample processing (cell lysis, DNA amplification and transcription) with a rapid and sensitive method, RPA.
 +
</p>
 +
</td>
 +
</tr>
 +
 +
<tr><td colspan=6 align=center valign=center>
 +
<h3>Readout circuit</h3>
 +
<p>
 +
We chose Cas13a for pathogen identification because of its specificity for nucleic acid sequence detection.
 +
</p>
 +
  <ul class="listResults">
 +
          <li>We characterized Cas13a and its detection limit with native and lyophilized protein, with in vitro and in vivo sources of RNA, in bulk and on paper. </li>
 +
          <li>We designed fluorescent and colorimetric readouts, and used a synthetic aptamer as a detection tool.
 +
</li>
 +
          <li>We tested targets from common pathogens and showed the orthogonality of virus detection versus bacterial detection. </li> 
 +
  </ul>
 +
<p>
 +
We proved the robustness and universality of our Cas13a-based fluorescence readout circuit.
 +
</p>
 +
</td>
 +
</tr>
 +
 +
<tr><td colspan=6 align=center valign=center>
 +
<h3>Readout circuit</h3>
 +
<p>
 +
We chose a disposable paper strip combined with a reusable fluorescence detector to analyse our samples.
 +
</p>
 +
  <ul class="listResults">
 +
          <li>We optimized the paper support for Cas13a functionality.</li>
 +
          <li>We built the most sensitive and cheapest fluorescence detector ever created by an iGEM team to our knowledge.
 +
</li>
 +
          <li>We successfully used our fluorescence detector to detect Cas13a activity on paper.</li> 
 +
  </ul>
 +
<p>
 +
We created a detection chip that is portable, functional and affordable, for the distribution of our diagnosis device, CascAID.
 +
</p>
 
</td>
 
</td>
 
</tr>
 
</tr>

Revision as of 22:39, 1 November 2017

Description

Design

Demonstrate

Measurement

Applied Design

Final Results

Achievements

Protocols

Notebook

Safety

Parts

Improved Part

Part Collection

InterLab

Model

Software

Collaboration

Detector

Quake Valve

Paperstrips

Sample Processing

Silver

Gold

Engagement

Collaborations

Team members

Sponsors

Attributions

Gallery

Demonstrate

With our modular approach, we prototype all units of our diagnostic device, and integrate them into a customizable platform. We use cell-free synthetic biology to distinguish between pathogens with our universal detection cascade.

Sensitive and Rapid

Robust and Universal

Portable and low-cost

Sample processing

We chose to combine heat lysis and isothermal amplification (RPA) to extract our target RNA from patient samples.

  • We used RPA to amplify DNA from heat lysed E. coli.
  • We conducted RPA and transcription from an in vitro DNA on paper.
  • We amplified and transcribed an in vitro DNA target to RNA concentrations detectable by our readout circuit .

We validated the three necessary modules of the sample processing (cell lysis, DNA amplification and transcription) with a rapid and sensitive method, RPA.

Readout circuit

We chose Cas13a for pathogen identification because of its specificity for nucleic acid sequence detection.

  • We characterized Cas13a and its detection limit with native and lyophilized protein, with in vitro and in vivo sources of RNA, in bulk and on paper.
  • We designed fluorescent and colorimetric readouts, and used a synthetic aptamer as a detection tool.
  • We tested targets from common pathogens and showed the orthogonality of virus detection versus bacterial detection.

We proved the robustness and universality of our Cas13a-based fluorescence readout circuit.

Readout circuit

We chose a disposable paper strip combined with a reusable fluorescence detector to analyse our samples.

  • We optimized the paper support for Cas13a functionality.
  • We built the most sensitive and cheapest fluorescence detector ever created by an iGEM team to our knowledge.
  • We successfully used our fluorescence detector to detect Cas13a activity on paper.

We created a detection chip that is portable, functional and affordable, for the distribution of our diagnosis device, CascAID.