From in silico to in vitro

As soon as the software code was ready to be used, we contacted professionals working in differenty labs and asked for opinions on what kind of toehold would make an impact on today's world. We brainstormed the list of possible viruses we could detect with our tool and came to the conclusion that Hepatitis C would be our new target, as it affects an estimated 71 million people1 globally and is found everywhere on the planet. An infection with Hepatitis C affects the liver and can lead to severe cirrhosis and liver cancer.

The disease is conventionally detected by screening for Hepatitis C virus (HCV) antibodies in the blood. This result is not conclusive as 15% to 45% of all infected people can get rid of the disease by themselves. Our detection scheme would provide people with early detection as it is not reliant on antibodies to develop, and it would yield an instant, non-ambiguous result. To put our software to the test, we gave it as an input a 251 base pairs-long genomic sequence unique2 to HCV genotype 1, which is the most abundant type of Hepatitis C in Central Africa, Europe and North America3.

Figure 1: Position of unique trigger sequences within the HCV genotype 1 sequence

After running the sequence with our software, we got as an output the best matching trigger/toehold pairs according to a scoring function. We ordered the four best scoring trigger/toehold pairs to test them in vitro, which we subsequently named A-D according to their position on the HCV genome (see Figure 1).

The toehold sequence is followed by the coding sequence of LacZ alpha, a small subunit of the LacZ gene. Each toehold is triggered by a unique trigger sequence (the trigger sequence is identical to a portion of the target genome, see Figure 1). After preparing the usual lysate reaction and following the results on the plate reader (please refer to our lysate reaction protocol), we were delighted to find that all the toeholds got triggered by their individual triggers and we had the hoped for result - and demonstrated that our software works!

A quick recap on our detection scheme : A toehold switch is an RNA molecule containing the toehold sequence and a downstream reporter gene. The toehold sequence makes the molecule fold into a hairpin structure, inhibiting translation of the reporter gene. The toehold sequence however will unfold to a linear form upon binding of its unique trigger.
We should only see translation of the reporter gene if the trigger is present in the same reaction. In presence of a viral trigger (for example from the blood sample of a person having the virus), we will produce beta-galactosidase (the protein form of LacZ), which will show by a change in colour. This occurs because beta-galactosidase's substrate changes its colour from yellow to purple after the enzymatic reaction of beta-galactosidase.

Figure 2: Toehold A in the iGEM backbone psB1C3

We performed the usual toehold reaction with those new toeholds : 10 uL reactions, measuring absorbance at 595nm on a plate reader, at 37°C. Those reactions were all set up as follows : 2.5 uL Energy Solution, 2.5 uL Buffer A, 2.5 uL lysate mix (T7 M15 + Top10-GamS), as well as toehold (final concentration 4 nM), trigger (final concentration 1 uM), substrate and water.

Figure 3: Kinetic measurements of the 4 generated toeholds in a usual lysate reaction
Shaded error graphs
Figure 4: End point measurements of the 4 generated toeholds in a usual lysate reaction

All the toeholds A, B, C, and D, led to a colour change visible by eye. For detailed results, please refer to this page.