Toehold Switch

Description

We decided to use a toehold switch system as a method to simulate RNA-dependent response in our cells. The image below briefly describes how this system works:

[Toehold]

In our system, the Trigger RNA represents an overexpressed messenger RNA which could be, for instance, a bacterial transport system related transcript, only expressed when the cell is under certain stress conditions and aimed to increase bacterial load. On the other hand, the Switch RNA represents the part capable of producing the desired effector, which would then be able to eliminate the related pathogen.

Firstly, we’ve carried out some experiments in order to evaluate the possibility to use this system in our work. For this purpose, two plasmids containing compatible origins of replications and adequate copy number were designed. pETDuet and pRSFDuet plasmids were chosen for the Switch and the Trigger RNA, respectively.

[Switch] [Trigger]

Results

Our team has successfully demonstrated the RNA-dependent response of our cells using a toehold switch circuit.

[Toehold2]

We then decided to assess if this response is IPTG-dependent, in an attempt to evaluate how much RNA is needed to activate the system:

[RNA1] [RNA2]

Iron and Lactate Biosensor

References

• Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell 159, 925–939 (2014).