Results

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 endogenous control sequence (EC sequence). The kit we used is DNeasy Plant Pro Kit by Qiagen.

DNA Extraction:
Here we performed Nanodrop UV absorption spectra :
1st graph. Red Line corresponds to gBlock;
Black Line corresponds to the Micro Needle extraction.

2nd graph. Green Line corresponds to gBlock;
Red Line corresponds to the Micro Needle extraction;
Blue Line corresponds to control (no DNA).

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 sequence 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 coumpounds 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:
Referred to Green et al. 2014 paper and optimized based on BioBits^TM toehold, 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, our desired length is 961 bps 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