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Revision as of 23:04, 29 October 2017

BioTROJAN Team
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CONCEPTUAL
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Overview Modeling Chassis BioContainment Detection Elimination
RESULTS
Overview Modeling Chassis BioContainment Detection Elimination Proof of Concept
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PRACTICES
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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:

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.

Results

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

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:

As shown by the graph above, IPTG concentrations as low as 0,01mM could activate the system! To assess if the observed expression was mostly related to the interaction between the trigger and the switch RNAs (and not due to some leakage of the Switch RNA), we compared the fluorescence profiles of a strain transformed with the two plasmids and another transformed with both a plasmid containing the Switch RNA and an empty plasmid.

Our last question was: would this system be sensitive enough to work under real conditions? In other words, mRNA expression variations observed in bacteria are really able to promote the activation of the Switch RNA? For this purpose, we decided to do some modeling, which you can check it out here!

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).