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| FD = Flavescence Dorée </br> | | FD = Flavescence Dorée </br> |
| BN = Bois Noir </br> | | BN = Bois Noir </br> |
− | EC = Endogeneous Control </br> | + | EC = Endogeneous Control (DNA from grapevines) </br> |
| </p> | | </p> |
| </div> | | </div> |
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| </div> | | </div> |
− | <div class="post-content"><p style="font-size:17px;" align="justify"> | + | <div class="post-content"> |
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− | <p style="font-size:17px;" align="justify">
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− | <br/>To test our method of extraction, we decided to try it on a non-infected grapevine leaf. To detect the extraction product, we performed a PCR We compared it to a traditional kit-based extraction, and to our synthetic endogeneous control sequence (EC sequence). The kit we used is DNeasy Plant Pro Kit by Qiagen. </br> | + | <p style="font-size:17px;" align="justify"> |
| + | To test our method of extraction, we used non-infected grapevine leaf. |
| + | To detect the product of the extraction, we performed an amplification by PCR. We compared the extraction by microneedles |
| + | to a traditional kit-based extraction. The control is synthetic endogeneous control DNA sequence (EC sequence). |
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| <br/> | | <br/> |
| + | </p> |
| + | <p style="font-size:17px" align="justify"><h7 style="color:purple"><b>PCR Amplification</b></h7></br> |
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| + | We amplified different EC DNA by PCR and ran a gel electrophoresis to analyze the results. |
| + | Bands are observed in all the lanes containing DNA, no band is present in the control. This shows that the |
| + | microneedle patch successfully extracted DNA from the plant, which was then amplified by PCR. |
| </p> | | </p> |
| <img src="https://static.igem.org/mediawiki/2019/d/de/T--EPFL--PCR_MN_EC.jpg" > | | <img src="https://static.igem.org/mediawiki/2019/d/de/T--EPFL--PCR_MN_EC.jpg" > |
− | <p style="font-size:17px;" align="justify">
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− | <br/><big>DNA Extraction:</big>
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− | <br/>Here we performed a Nanodrop UV absorption spectra with the DNA extracted with the micro needle patches and an endogeneous control : <br> The red Line corresponds to the endogeneous control (EC); <br> The black Line corresponds to the Micro Needle extraction. <br>
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− | <img src="https://static.igem.org/mediawiki/2019/3/35/T--EPFL--2courbes2.png" >
| + | <p style="font-size:17px;" align="justify"></br><h7 style="color:purple"><b>Nanodrop Analysis</b></h7> |
− | <br/> 2nd graph. Green Line corresponds to gBlock; <br> Red Line corresponds to the Micro Needle extraction; <br> Blue Line corresponds to control (no DNA). | + | |
| + | <br/> We used the Nanodrop to analyze the DNA extracted with a microneedle patch (red). The positive control (green) is EC synthetic DNA sequence and the negative control (blue) is the elution buffer applied on an unused microneedle patch. </br> |
| + | As expected, the negative control absorption at 260nm is close to zero, no DNA is present in the microneedle patch. In contrast, the positive control has the typical DNA Nanodrop readout. </br> |
| + | The graph of DNA extracted by microneedle patch shows a peak of absorption at 260nm, we can also see that that values absorption values at 230nm and 280nm are lower that the value at 260nm. Overall, the graph is similar to the positive control but with lower values. This indicates that DNA was extracted by the microneedle patch. |
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| </p> | | </p> |
− | <img src="https://static.igem.org/mediawiki/2019/c/c9/T--EPFL--3courbes.png" > | + | <img src="https://static.igem.org/mediawiki/2019/2/22/T--EPFL--courbes.png" > |
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| </div> | | </div> |
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− | <div class="post-content"><p style="font-size:18px" align="justify"> </br> <h7 style="color:purple"><b>Multiplexing :</b></h7> <br> as our final test would contain all 3 primer pairs, we tested if the amplification was functional with various combinations of primer pairs. The results show that amplification is successful for each test, though the endogenous sequence seem to amplify more than the phytoplasma sequences.</p> | + | <div class="post-content"><p style="font-size:18px" align="justify"> </br> <h7 style="color:purple"><b>Multiplexing</b></h7> |
| + | <br> As our final test would contain all 3 primer pairs, we tested if the amplification was functional with various |
| + | combinations of primer pairs. The results show that amplification is successful for each test, though the endogenous |
| + | sequences seem to amplify more than the phytoplasma sequences.</p> |
| <img src="https://static.igem.org/mediawiki/2019/c/c9/T--EPFL--multiplexing.jpg" height=auto width=auto > | | <img src="https://static.igem.org/mediawiki/2019/c/c9/T--EPFL--multiplexing.jpg" height=auto width=auto > |
| <br> | | <br> |
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| </br> | | </br> |
| <p style="font-size:18px" align="justify"> | | <p style="font-size:18px" align="justify"> |
− | <h7 style="color:purple"><b>Amplification in grapevine extract : </b></h7> | + | <h7 style="color:purple"><b>Amplification in grapevine extract </b></h7> |
− | We wanted to know if the RPA would be hindered by the presence of plant coumpounds extracted along with the DNA (in particular, phenols and polysaccharides are known to act as PCR inhibitors<a href="#section8"><sup>8</sup></a>). Using our microneedle method, we extracted the DNA of an uninfected grapevine leaf. We then carried out two experiments : | + | </br>We wanted to know if the RPA would be hindered by the presence of plant compounds extracted along with the DNA (in particular, phenols and polysaccharides are known to act as PCR inhibitors<a href="#section8"><sup>8</sup></a>). Using our microneedle method, we extracted the DNA of an uninfected grapevine leaf. We then carried out two experiments : |
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| <ul style="font-size:18px" > | | <ul style="font-size:18px" > |
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| </p> | | </p> |
| + | </br> |
| + | </br> |
| + | <p style="font-size:17px; align="justify"> |
| + | <h7 style="color:purple"><b>Transcription </b></h7> |
| + | </br> |
| + | </br> |
| + | To test that our amplicons could indeed be transcribed, we did an <i>in vitro</i> transcription reaction using HiScribe™ T7 Quick High Yield RNA Synthesis Kit. The transcription product were detected by electrophoresis on a 2% denaturing agarose gel. |
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| + | </p> |
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| + | <img src="https://static.igem.org/mediawiki/2019/9/9a/T--EPFL--transcription_rpa.jpg" height=auto width=auto > |
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| + | <p style="font-size:13px"><i> Figure 12 : Agarose gel electrophoresis of post-RPA transcription product</i></p> |
| + | <p style="font-size:17px; align="justify"> |
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| + | The transcription of all 3 RPA products (lanes 1, 4 and 7) is similar to that or their synthetic counterpart sequences with T7 (lanes 2, 5 and 8). This means that RPA was successful in adding a functional T7 promoter to the amplified sequences. |
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| + | </p> |
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| </div> | | </div> |
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| <div class="post-content"> | | <div class="post-content"> |
| <p style="font-size:17px;" align="justify"> | | <p style="font-size:17px;" align="justify"> |
− | <br/><h7 style="color:purple"><b>Toehold design:</b></h7> | + | <br/><h7 style="color:purple"><b>Toehold design</b></h7> |
− | <br/>Referred to <i>Green et al. 2014</i> paper and optimized based on BioBits<sup>TM</sup> toehold, we designed the following toeholds. Each group has 4 candidates who ranked as top 4 in their design score. | + | <br/>Referring to the <i>Green et al. 2014</i> paper and optimized based on the BioBits<sup>TM</sup> toehold design, we designed the following toeholds. Each group has 4 candidates who ranked as top 4 in their design score. |
| </p> | | </p> |
| <img src="https://static.igem.org/mediawiki/2019/6/68/T--EPFL--results_toehold_design1.png" > | | <img src="https://static.igem.org/mediawiki/2019/6/68/T--EPFL--results_toehold_design1.png" > |
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| <img src="https://static.igem.org/mediawiki/2019/2/2f/T--EPFL--results_toehold_design3.png" > | | <img src="https://static.igem.org/mediawiki/2019/2/2f/T--EPFL--results_toehold_design3.png" > |
| <p style="font-size:17px;" align="justify"> | | <p style="font-size:17px;" align="justify"> |
− | <br/><h7 style="color:purple"><b>Toehold assembly :</b></h7> | + | <br/><h7 style="color:purple"><b>Toehold assembly</b></h7> |
− | <br/>Here we take BN 2.1 (Bois Noir 2<sup>nd</sup> Version, N°1) toehold as an example, our desired length is 961 bps which is approved by our Electrophoresis gel: | + | <br/>Here we take BN 2.1 (Bois Noir 2<sup>nd</sup> Version, N°1) toehold as an example, so our desired length is 961 bp which is confirmed by our Electrophoresis gel: |
| <br/> | | <br/> |
| </p> | | </p> |
| <img src="https://static.igem.org/mediawiki/2019/2/24/T--EPFL--results_toehold_assembly1.png" > | | <img src="https://static.igem.org/mediawiki/2019/2/24/T--EPFL--results_toehold_assembly1.png" > |
| <p style="font-size:17px;" align="justify"> | | <p style="font-size:17px;" align="justify"> |
− | <br/><h7 style="color:purple"><b>Toehold functionality:</b></h7> | + | <br/><h7 style="color:purple"><b>Toehold functionality</b></h7> |
| </p> | | </p> |
| <p style="font-size:17px;" align="justify"> | | <p style="font-size:17px;" align="justify"> |