We designed our first generation of toeholds based on the Green et al. 2014 paper.
As the trigger mRNA will be the amplicon from our RPA product (approx. 70 bp), the trigger
binding region is designed to contain the complimentary nucleotides. As described in the paper,
one of the optimal sizes of the trigger binding region is 36 nts. Based on that number, we
designed a library of approximately 35 toeholds for each disease and control. In order to
keep the hairpin structure stable when it is deactivated, we calculated its Normalized Ensemble
Defect (NED, The average percentage of nucleotides that are incorrectly paired at equilibrium
relative to the specified secondary structure, evaluated over the Boltzmann-weighted ensemble of
(unpseudoknotted) secondary structures (0% is best, 100% is worst)) using the NUPACK software
suite. All the toeholds were then evaluated using the following equation provided by the paper and
ranked accordingly (0 is best, 100 is worst):
where ϕ is the design score for the sensor at location i of the mRNA.
- lmRNA is the local single-strandedness (calculated as NED) of the mRNA at the sensor binding site.
- ltoehold is the local single-strandedness of the toehold of the sensor.
- nsensor is the NED of the sensor.
- The score weight factors used were β1 = 5, β2 = 4, and β3 = 3.
Only the top 4 toeholds were selected and carried on to the next step.
In order to monitor the kinetics of our detection using toehold regulation, we chose
sfGFP as the reporter gene and therefore connected our toeholds to an sfGFP
sequence. We ordered forward primers for sfGFP, each containing a toehold as overhang and
a common reverse primer. To assemble it we simply performed a PCR using the iGEM distributed
sfGFP plasmid as template. You can check the protocol here.
To check if the products matched our desired sequence, we performed a gel electrophoresis followed by Sanger sequencing to confirm the results.
We have a higher sfGFP expression rate than our reference set result, but we still got
a low leakage at the OFF state.
The functionality test was then performed in both OnePot PURE and the NEB commercial
PURExpress system, using ssDNA ordered from IDT as triggers to limit undesired factors
(RNase contamination, DNA transcription, etc...). For each test, we ran a 5 μl reaction in
a 384-well-plate, then recorded the GFP absorbance in a microplate reader at 37°C for over
2 hours. You can check the protocol here.
CDO was expressed in OnePot PURE with 1mM of catechol and incubated at 37°C. You can check our protocol here.
To be sure that the change of color was the result of the cleavage of catechol by CDO we
made two controls. The first was to express CDO into OnePot PURE without catechol, the second
was to add catechol without CDO.