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)

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 amplified different EC DNA by PCR and ran a gel electrophoresis to analyze the results. We loaded the gel from left to right as following, the ladder, synthetic EC DNA sequence, DNA extracted with a microneedle patch, DNA extracted with a standard kit, control with primers but no DNA. 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.

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 DNA extracted by microneedle patch shows significant absorption at A260. Its line higher than the negative control but lower than the positive control. This means that some DNA was extracted but that it is not pure, which is normal since, as mentioned previously, the patch extracts DNA and other molecules on the leaf.

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:

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.