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| − | <h2 style="color:purple" ><br> <br> <br> <br> Results</h2> | + | <h2 style="color:purple" ><br> <br> <br> <br> Results</h2> |
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| − | <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 : | 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 : | ||
| − | <ul style="font-size:18px" > | + | <ul style="font-size:18px" > |
<li> We tested that our RPA worked for endogenous control in plant extract</li> | <li> We tested that our RPA worked for endogenous control in plant extract</li> | ||
| − | <li> We performed a limit of detection by spiking different concentrations of our synthetic FD DNA into the microneedle extract (MNE) </li> | + | <li> We performed a limit of detection by spiking different concentrations of our synthetic FD DNA into the microneedle extract (MNE) </li> |
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<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, our desired length is 961 bps which is approved by our Electrophoresis gel: |
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But in the real case, we will use dsDNA as trigger, thus it is essential to compare their expression rates: | But in the real case, we will use dsDNA as trigger, thus it is essential to compare their expression rates: | ||
</p> | </p> | ||
| − | <img src="https://static.igem.org/mediawiki/2019/ | + | <img src="https://static.igem.org/mediawiki/2019/d/d9/T--EPFL--Detection_toehold_result_1.png" > |
<p style="font-size:17px;" align="justify"> | <p style="font-size:17px;" align="justify"> | ||
The error bar of the expression rate overlaps, no significant difference is detected.<br/><br/> | The error bar of the expression rate overlaps, no significant difference is detected.<br/><br/> | ||
In order to test the limit of detection of our toehold, we've run a test for toehold expression in different concentration of trigger DNA, by theory in the detectable range the difference should be bigger when the concentration of trigger increases: | In order to test the limit of detection of our toehold, we've run a test for toehold expression in different concentration of trigger DNA, by theory in the detectable range the difference should be bigger when the concentration of trigger increases: | ||
</p> | </p> | ||
| − | <img src="https://static.igem.org/mediawiki/2019/ | + | <img src="https://static.igem.org/mediawiki/2019/f/f8/T--EPFL--Detection_toehold_result_2.png" > |
<p style="font-size:17px;" align="justify"> | <p style="font-size:17px;" align="justify"> | ||
There is a detectable difference from 100nM, and it grows when the concentration goes up, which suits our theory. | There is a detectable difference from 100nM, and it grows when the concentration goes up, which suits our theory. | ||
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<img src="https://static.igem.org/mediawiki/2019/7/75/T--EPFL--resultsOnePot5.png" > | <img src="https://static.igem.org/mediawiki/2019/7/75/T--EPFL--resultsOnePot5.png" > | ||
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</center> | </center> | ||
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</div> | </div> | ||
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 EC sequence. The kit we used is DNeasy Plant Pro Kit by Qiagen.
DNA Extraction:
Here we performed Nanodrop UV absorption spectra : Red Line corresponds to gBlock;
Black Line corresponds to MN extraction.
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.
The endogenous control amplification was successful in MNE.
The limit of detection seems to show bands for FD as low as 10 copies/μl (50 copies total). We can see a "ladder pattern" for concentrations equal to or below 1000 copies/μl. This pattern occurs when the concentration of template is too low and unspecific primer-driven amplification happens (See the DNA amplification page for more details).
All in all, RPA has proved to function in grapevine extract.
Referred to Green et al. 2014 paper and optimized based on BioBitsTM toehold, we designed the following toeholds. Each group has 4 candidates who ranked as top 4 in their design score.
Here we take BN 2.1 (Bois Noir 2nd Version, N°1) toehold as an example, our desired length is 961 bps which is approved by our Electrophoresis gel:
In our project, we've used two kind of triggers. For the ssDNA trigger, there is no need of T7 polymerase transcription, therefore eliminate some uncertainties. But in the real case, we will use dsDNA as trigger, thus it is essential to compare their expression rates:
The error bar of the expression rate overlaps, no significant difference is detected.
In order to test the limit of detection of our toehold, we've run a test for toehold expression in different concentration of trigger DNA, by theory in the detectable range the difference should be bigger when the concentration of trigger increases:
There is a detectable difference from 100nM, and it grows when the concentration goes up, which suits our theory.
The DNA sequence coding for catechol-2,3-deoxygenase (CDO), and completed with an ribosome binding site (rbs) and T7 promoter and terminator sites, was successfully assembled from the XylE (gene coding for CDO) template provided in the iGEM 2019 DNA Distribution kit, by using a 2-step PCR protocol. The gene assembly was verified by a Sanger DNA sequencing which showed that the DNA template was 99.8% accurate, for a total sequence length of 1045 bases.
This sequence was then expressed in our OnePot PURE cell-free system and incubated in presence of catechol. A yellow color was observed after 30 minutes of incubation, and it became brighter one hour after the start of the reaction. There were no colors in the t wo controls performed, one without catechol but with CDO template and the other one without CDO template but with catechol. This proved that the color was indeed created by the reaction of CDO with catechol and not by self-oxidation of catechol.
Expression of sf GFP on OnePot and PURExpress for 5nM concentration of the DNA template, measured in the plate reader using excitation wavelength of 535nm .
