Revision as of 22:01, 21 October 2019

Results

During our Experiments we used abreviations to simplify the results annotation.
MN = Microneedle
FD = Flavescence Dorée
BN = Bois Noir
EC = Endogeneous Control

DNA Extraction

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.

DNA Extraction:
We performed a Nanodrop UV absorption spectra with the DNA extracted with the microneedle patches, an endegeneous control, and elution buffer applied on an unused microneedle patch as a control.
The green Line corresponds to the EC;
The red Line corresponds to the Microneedle extraction;
The blue Line corresponds to the elution buffer applied on an unused microneedle patch (no DNA).
The DNA concentration is high enough to have an apparent signal on the graphs (bump on the curve). We can see that there is no signal for the control as the curve is completely flat and close to zero, however we have DNA extracted with our MN patches.

DNA Amplification

Multiplexing
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.

Amplification in grapevine extract
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 inhibitors⁸). Using our microneedle method, we extracted the DNA of an uninfected grapevine leaf. We then carried out two experiments :

We tested that our RPA worked for endogenous control in plant extract
We performed a limit of detection by spiking different concentrations of our synthetic FD DNA into the microneedle extract (MNE)

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.

Toehold switches

Toehold design
Referring to the Green et al. 2014 paper and optimized based on the BioBits^TM toehold design, we designed the following toeholds. Each group has 4 candidates who ranked as top 4 in their design score.

Toehold assembly
Here we take BN 2.1 (Bois Noir 2^nd Version, N°1) toehold as an example, so our desired length is 961 bp which is approved by our Electrophoresis gel:

Toehold functionality

We used a commercially available toehold sensor (pCOLA_banana_sfGFP_sensor, BioBits^TM), and expressed it in NEB PURExpress^TM PURE system as our reference expression of a toehold. We compared it with our BN 2.1 toehold expressed in our OnePot PURE system in the figure below:

We also compared the ON/OFF ratio and the leakage between these two systems:

Signal Generation

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.

OnePot PURE

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 .

@@ Line 139: / Line 139: @@
 <p style="font-size:17px;" align="justify">
          <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, so 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 approved by our Electrophoresis gel:
          <br/>
 </p>