Our headline is simple: we managed to reproduce the RNA switch described for Zika virus! But how do you get from zero to a functional laboratory workflow?
One key issue is organization: we divided our science team in specialists for DNA sequence design, cell-free expression and analytics respectively. The second key point is a procedure with several checkpoints, so if ever there are complications - which in science is practically always the case - we are sure to know in what part of the process they arise.
Thus, in a first step, the team specializing in cell-free systems expressed a fluorescent protein, to control for functionality of the setup. As this worked reliably, they moved on to expressing both RNA switch and the RNA sequence triggering the switch, using the information from the Zika publication. Simultaneously, the DNA team produced the beta-galactosidase necessary for the color reaction in combination with a T7 promotor sequence. This promoter sequence is derived from a bacteriophage and is extensively used in molecular biology, as it ensures high expression of the desired gene.
The RNA switch, the triggering RNA sequence as well as the beta-galactosidase were then combinedly expressed in our cell-free expression chambers - and as you can see in the image above, it worked perfectly: The three samples on the left contain the trigger RNA and are thus purple, while two control samples on the right remain yellow, as we expected.
With this encouraging result, we are currently trying to get this testing setup closer to our actual goal: How can we fix the cell-free expression system on a paper strip? How can we make the color reaction brighter? How make it efficient at any temperature? ... There's much to be investigated, and we hope to be able to answer some of these questions soon!
(1) Original publication for Zika 27B switch: http://www.cell.com/fulltext/S0092-8674(16)30505-0