Toehold experiments

In order to show that toehold switches could be developed to be used in our diagnostic test, we first had to show that the toehold can be expressed cell-free, and that it responds to the correct trigger RNA. To do this we have tested two toehold designs; Green_FET1, which was designed and tested by Green et al., and our own toehold design, code-named Zeus. Before testing these designs in the lab, which is expensive due to the high cost of commercial cell-free kits, we decided to test the toeholds in silico using the online software package NUPACK.The use of the control toehold, Green_FET1, which has previously been reported to work as expected experimentally, allows for comparison of in silico between our toehold (Zeus), and the data of a function toehold. This can then be used to allow for an informed decision as to whether the toehold will work practically.

In silico testing:

The use of in silico testing in synthetic biology, and indeed science as a whole, is something which is not to be overlooked. While practical experimentation is still required and an important aspect of any project, the correct use of this kind of testing and modelling can allow for better informed experiments and more efficient testing, which can be extremely important when testing may take long time, or, as in this project's case, become expensive if many experiments are run.

As our diagnostic test would be carried out in the field, where the temperature can not be easily controlled, it was important to determine the integrity of the toehold structures at different temperatures. In order to do this the analysis function of NUPACK was used to perform a melt between 0C and 70C on both toeholds. Briefly, the Green_FET1 toehold structure was shown to correct from 25C, and the Zeus toehold was shown to be correct from 15C. A full discussion of this result can be found on the results page.

Toehold dynamic range

Protocols:

Standard Operating Procedures (SOPs)

Use the arrow keys or touch screen to scroll through our SOPs. Click on an image to bring up the corresponding protocol.