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| − | <img id="TopPicture" width="960" src="https://static.igem.org/mediawiki/2017/b/be/T--Munich--FrontPagePictures_Attributions.jpg">
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| − | <tr><td colspan=6 align=left valign=center> | + | <tr id="hardwareFrontPage"> |
| − | <font size=7 color=#51a7f9><b style="color: #51a7f9">Introduction</b></font>
| + | <td colspan=2> |
| − | </td> | + | <table width=320> |
| − | </tr>
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| − | <tr>
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| − | <td colspan = 6 align="left">
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| − | <p class="introduction">
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| − | Our pathogen detection approach relies on Cas13a digesting RNA. A change in fluorescence is the most convenient way to take trace of such enzyme kinetics. This is impractical for in-field applications because commercial fluorescence detectors are expensive and inconveniently large. A straightforward approach for making our pathogen detection fit for in-field applications is a cheap and handy fluorescence detector. Other iGEM teams already constructed fluorescence detectors. What they all lack is a high sensitivity and the ability to measured fluorescence quantitatively.
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| − | We therefore constructed a detector matching our requirements. Our detector is paper-based and can detect fluorescein concentration down to 200 nM. The detector measures fluorescence in units of equivalent fluorescein concentrations. The detector is very compact and costs less than 10 EUR. We were able to measure a time trace of Cas13a digesting RNaseAlert with our detector. For comparison we also measured a positive control containing RNase A and a negative control containing only RNaseAlert. The data are displayed in image ~\ref{img:k3}. The time traces show an enzymatic reaction taking place on filter paper. This proves that our detector is sensitive enough and meets our requirements. However the detector is not limited to the detection of our specific application. It can be used for the detection of any fluorescence signal in biological or chemical systems. We think that our detector can benefit other iGEM teams and also research groups that want to make a fluorescence based detection fit for in-field applications.
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| − | </p>
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| − | </td>
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| − | <tr><td colspan=6 align=center valign=center>
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| − | <h3>CascAID</h3>
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| − | <p>
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| − | Our project, which we named Cas13a controlled assay for infectious diseases (CascAID), features the recently identified CRISPR/Cas effector Cas13a<sup><a class="myLink" href="#ref_10">10</a></sup>. Unlike other proteins in the familiy, Cas13a has the unique ability to bind and cleave specific RNA targets rather than DNA ones. Moreover, after cleaving its target, Cas13a is able to unspecifically cleave RNA molecules. By using this collateral activity from Cas13a, our system is capable of detecting virtually any RNA target. This is done by changing the crRNA in the protein, that is a short RNA sequence that determines what is recognized as target.</p>
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| − | </td>
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| − | </tr>
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| | <tr> | | <tr> |
| − | <td colspan=6 align=center valign=center> | + | <td> |
| − | <img src="https://static.igem.org/mediawiki/2017/0/04/T--Munich--Description_Cas13a_Mechanism.svg" alt="Diagram for Cas13a's function"> | + | <a href="/Team:Munich/Hardware/QuakeValve"><img class="picture1" src="https://static.igem.org/mediawiki/2017/0/ 08/T--Munich-- Overlay. png"></ a> |
| − | <p>Cas13a binds specific target RNA depending on the crRNA sequence. After activation, Cas13a cleaves RNA indiscriminately.</p>
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| | </td> | | </td> |
| − | | + | <td> |
| − | </tr>
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| − | | + | |
| − | <tr><td align=center valign=center colspan=4>
| + | |
| − | <p>
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| − | We wanted to start our project by showing that Cas13a's collateral activity could be used to detect the presence of specific RNA. For this, we used the RNAse alert system, as done in a recent publication<sup><a class="myLink" href="#ref_11">11</a></sup>, to detect RNA digestion. In this assay, the presence of RNAse-like activity is detected by an increase in green fluorescence. Our experiments yielded a convincing proof-of-principle which we went on to model. Moreover, CascAID can be used to detect a wide spectrum of pathogens, as our experiments with gram-positive and viral targets suggested. As we wanted to make CascAID available for everyone, we focused on building an inexpensive fluorescence detector to measure the presence of the target. Our detector “Lightbringer” was designed to be able to detect the fluorescence produced by the fluorescein in the Rnase alert system<sup><a class="myLink" href="#ref_12">12</a></sup>, but we theorize that changing the filters allows detection of other fluorophores. In addition, we experimented with freeze-drying on paper to make CascAID durable and easily portable.
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| − | </p>
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| | </td> | | </td> |
| − | <td align=center valign=center colspan=2> | + | </tr> |
| − | <img src="https://static.igem.org/mediawiki/2017/7/7f/T--Munich--Description_Cas13a_Readout_Comparision.svg">
| + | </table> |
| − | <p>Cas13a can be used to detect specific RNA sequences</p>
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| − | </td>
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| − | </tr>
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| − | <tr class="lastRow"> | + | <a href="/Team:Munich/Hardware/SampleProcessing"><img id="picture3" src="https://static.igem.org/mediawiki/2017/0/08/T--Munich--Overlay.png"></a> |
| − | <td align=center valign=center colspan=2> | + | <a href="/Team:Munich/Hardware/Paperstrip"><img id="picture2" src="https://static.igem.org/mediawiki/2017/ 0/ 08/T--Munich-- Overlay. png"></a> |
| − | <a href=" http:// www. uni-muenchen. de/ studium/ lehre_at_lmu/ index. html"><img src="https://static.igem.org/mediawiki/2017/ 9/ 9a/T--Munich-- Logo_LehreLMU. gif" width="200"></a > | + | |
| − | <p>Picture of the Thermocycler</p>
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| | </td> | | </td> |
| − | <td align=center valign=center colspan=4> | + | <td colspan=4> |
| − | <p> | + | <a href="/Team:Munich/Hardware/Detector"><img id="picture4" src="https://static.igem.org/mediawiki/2017/0/08/T--Munich--Overlay.png"></a> |
| − | For RNA extraction from the samples we tested three methods: extraction with silica beads, extraction with silica membrane and heat lysis. We custom-built an affordable thermocycler for signal amplification by RT-PCR to improve the detection limit. We explored recombinase polymerase amplification (RPA), an isothermal amplification procedure, to use over more conventional PCR methods as its simplicity makes it the more attractive option.
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| − | </p>
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| − | </td>
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| − | </tr> | + | |
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| − | <tr><td colspan=6 align=center valign=center>
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| − | <h3>Colorimetric read-outs</h3>
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| − | <p>
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| − | To couple CascAID with an easy read-out method we explored three colorimetric read-outs:
| + | |
| − | </p> | + | |
| | </td> | | </td> |
| | </tr> | | </tr> |
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| − | <tr><td colspan=2 align=center valign=center> | + | <tr> |
| − | <p> | + | <td colspan=6> |
| − | <b>AeBlue</b>: The RNA strand in a specially designed RNA/DNA dimer is cut by Cas13a's collateral
| + | <div class="captionPicture"> |
| − | activity. After digestion, the interaction between the two strands is too weak to hold the dimer and it
| + | <p> |
| − | decays. We can then use the DNA-strand as template to translate the chromoprotein <a href="http://parts.igem.org/Part:BBa_K864401">aeBlue</a>.
| + | Complete overview of all modular hardware parts in our pathogen detection system. Shown are counterclockwise and starting in in the upper left corner: The Quake valve that controls fluid flow, our sample processing device, the paper strip where a reaction mix is stored and the readout reaction takes place and finally our low-cost fluorescence detector "Lightbringer" that performs the readout measurement. Images are clickable and linked to the corresponding wiki subsection. |
| − | </p>
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| − | </td>
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| − | <td colspan=4 align=center valign=center>
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| − | <img src="https://static.igem.org/mediawiki/2017/9/90/T--Munich--Description_aeBlue.svg">
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| − | <p>Diagram of aeBlue</p>
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| − | </td>
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| − | </tr>
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| − | <tr><td colspan=2 align=center valign=center>
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| − | <p>
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| − | <b>Intein-Extein</b>: By binding TEV-protease with a RNA-linker we can use Cas13a's collateral activity
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| − | to regulate the protease's diffusion and use it to cleave a TEV tag separating the intein regions of a
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| − | modified chromophore. After the first cleavage, the intein segment excises itself<sup><a class="myLink" href="#13">13</a></sup>, bringing together the
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| − | halves of the chromophore. Only then is the chromophore functional and produces the colorimetric
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| − | read-out.
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| | </p> | | </p> |
| − | </td> | + | </div></td> |
| − | <td colspan=4 align=center valign=center>
| + | </tr> |
| − | <a href="http://www.uni-muenchen.de/studium/lehre_at_lmu/index.html"><img src="https://static.igem.org/mediawiki/2017/9/9a/T--Munich--Logo_LehreLMU.gif" width="200"></a>
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| − | <p>Diagram of Intein-Extein</p>
| + | |
| − | </td> | + | |
| − | </tr> | + | |
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| − | <tr class="lastRow"><td colspan=2 align=center valign=center> | + | <tr><td colspan=6 align=left valign=center> |
| − | <p> | + | <font size=7 color=#51a7f9><b style="color: #51a7f9">Hardware</b></font> |
| − | <b>Gold nanoparticles</b>: Gold nanoparticles coated with short DNA sequences are held closely
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| − | together by a complementary linker RNA, which makes the solution intense blue<sup><a class="myLink" href="#14">14</a></sup>. Activated Cas13a cuts
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| − | the linker RNA, causing the nanoparticles to diffuse away from each other. This increase in distance
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| − | causes a color change to intense red.
| + | |
| − | </p>
| + | |
| − | </td>
| + | |
| − | <td colspan=4 align=center valign=center>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/b/b3/T--Munich--Description_Goldnanoparticles.svg">
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| − | <p>Gold nanoparticles</p>
| + | |
| − | </td>
| + | |
| − | </tr>
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| − | | + | |
| − | <tr><td colspan=6 align=center valign=center>
| + | |
| − | <h3>Software</h3>
| + | |
| − | <p>
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| − | To help facilitate the design of crRNA, the sequences that give CascAID its specificity, we developed a
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| − | software tool that checks crRNA for unwanted secondary structures. This gives valuable insight on
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| − | whether the sequence is suited to use with Cas13a or whether some modifications are needed.
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| − | Together with Team Delft's software tool which designs the corresponding crRNA based on the target,
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| − | we collaborated to develop a powerful tool that suggests crRNA sequences and checks their usability
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| − | </p> | + | |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | + | <tr> |
| | + | <td colspan = 6 align=center valign=center> |
| | + | <p class="introduction"> |
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| | + | The liberation of diagnostic tests from expensive lab infrastructure requires innovative ways of sample processing and measuring. We therefore developed a set of portable hardware tools with the goal of providing an automated sample-to-answer solution. The heart of our system is <a class="myLink" href="https://2017.igem.org/Team:Munich/Hardware/Detector">‘Lightbringer’</a>, our fluorescence detector, which is capable of measuring kinetics of biological or chemical reactions on <a class="myLink" href= "https://2017.igem.org/Team:Munich/Hardware/Paperstrip" >paper.</a> Built from 3D–printed parts and standard electronic components, it can be assembled for less than 15$, while offering a sensitivity competitive to commercial fluorescence readers. Additionally, tackling the challenge of sample pre-processing in field, we developed a portable <a class="myLink" href="https://2017.igem.org/Team:Munich/Hardware/SampleProcessing"> fluidic system</a>, featuring a temperature control unit for lysis and isothermal PCR. Conceiving a platform independent of lab infrastructure, we demonstrate the feasibility of <a class="myLink" href="https://2017.igem.org/Team:Munich/Hardware/QuakeValve"> controlling fluid flow</a> with bike tires and air balloons. All hardware components are designed and documented with the aim of enabling the community to reproduce and extend our set of tools. |
| | + | </p> |
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| − | <tr><td colspan=6 align=center valign=center> | + | <div class="captionPicture"> |
| − | <h3>References</h3>
| + | <img width=960 src="https://static.igem.org/mediawiki/2017/2/26/Schema_final_lowres.png"> |
| − | <p>
| + | <p> |
| − | <ol style="text-align: left">
| + | |
| − | <li id="ref_1">Cohen, Limor, and David R. Walt. "Single-Molecule Arrays for Protein and Nucleic Acid Analysis." Annual Review of Analytical Chemistry 0 (2017).</li>
| + | |
| − | <li id="ref_2">Nakano, Michihiko, et al. "Single-molecule PCR using water-in-oil emulsion." Journal of biotechnology 102.2 (2003): 117-124.</li>
| + | |
| − | <li id="ref_3">Taniguchi, Yuichi, et al. "Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells." science 329.5991 (2010): 533-538.</li>
| + | |
| − | <li id="ref_4">Rissin, David M., et al. "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations." Nature biotechnology 28.6 (2010): 595-599.</li>
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| − | <li id="ref_5">Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.</li>
| + | |
| − | <li id="ref_6">Slomovic, Shimyn, Keith Pardee, and James J. Collins. "Synthetic biology devices for in vitro and in vivo diagnostics." Proceedings of the National Academy of Sciences 112.47 (2015): 14429-14435.</li>
| + | |
| − | <li id="ref_7">Tang, Ruihua, et al. "A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection." Lab on a Chip 17.7 (2017): 1270-1279.</li>
| + | |
| − | <li id="ref_8">Vashist, Sandeep Kumar, et al. "Emerging technologies for next-generation point-of-care testing." Trends in biotechnology 33.11 (2015): 692-705.</li>
| + | |
| − | <li id="ref_9">Gubala, Vladimir, et al. "Point of care diagnostics: status and future." Analytical chemistry 84.2 (2011): 487-515.</li>
| + | |
| − | <li id="ref_10">Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353.6299 (2016): aaf5573.</li>
| + | |
| − | <li id="ref_11">Gootenberg, Jonathan S., et al. "Nucleic acid detection with CRISPR-Cas13a/C2c2." Science (2017): eaam9321.</li>
| + | |
| − | <li id="ref_12">https://www.idtdna.com/pages/docs/technical-reports/in_vitro_nuclease_detectionD325FDB69855.pdf (retrieved: 13.10.17)</li>
| + | |
| − | <li id="ref_13"> Anraku, Yasuhiro, Ryuta Mizutani, and Yoshinori Satow. "Protein splicing: its discovery and structural insight into novel chemical mechanisms." IUBMB life 57.8 (2005): 563-574.</li>
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| − | <li id="ref_14">Link, Stephan, and Mostafa A. El-Sayed. "Size and temperature dependence of the plasmon absorption of colloidal gold nanoparticles." The Journal of Physical Chemistry B 103.21 (1999): 4212-4217.</li>
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| − | </ol>
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