We divided the project into five separate modules: DNA extraction, DNA amplification, Toeholds, Signal Generation and OnePot PURE. We assigned sub-teams to each one of these modules. Each sub-team worked on making sure their module is functional on their own. Once each part worked independently, we started linking them to prove the functionality of our test.
Worked on testing and controlling the DNA extraction efficiency of two different methods for sample processing: 1) A physical and/or chemical DNA extraction disruption with sample clarification; 2) A microneedle patch for rapid DNA extraction. Both methods were compared using a spectrophotometer and gel electrophoresis.
Worked on amplifying low amounts of DNA to make it detectable. Our method is based on an isothermal variant of PCR called RPA (Recombinase Polymerase Amplification). We focused on amplifying specific double-stranded DNA sequences from the Flavescence Dorée, Bois Noir and grapevine genomes, which required us to design and test several primer pairs. After selecting the best of them, a T7 promoter was added to the primer sequences to enable transcription during the detection step.
Worked on designing and constructing novel toehold switches that are compatible with the amplicons generated by the amplification step. RPA creates amplicons using Flavescence Dorée, Bois Noir, and the endogenous grapevine as templates. The optimization of the toehold structure was done using NUPACK to prevent any leakage.
Worked on testing whether the proteins GFP and CDO can be efficiently produced and detected using our OnePot PURE system. GFP creates a fluorescent signal, easily measured with a plate reader. CDO is an enzyme which, by reacting with the substrate catechol, creates a yellow color.
Worked on demonstrating that our homemade OnePot PURE cell-free system is functional. They provided data on the quality of our protein solution and our ribosome preparation.