Results

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

DNA Extraction

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).

PCR Amplification
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.

Nanodrop Analysis
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.
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.
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.

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.

Transcription

To test that our amplicons could indeed be transcribed, we did an in vitro transcription reaction using HiScribe™ T7 Quick High Yield RNA Synthesis Kit. The transcription product were detected by electrophoresis on a 2% denaturing agarose gel.

Figure 12 : Agarose gel electrophoresis of post-RPA transcription product

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.

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 confirmed 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