Team:USP-Brazil/Results/Detection
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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
Another way to detect diseases is through the detection of metabolites that are qualitatively or quantitatively altered in the specific condition. As a way of detecting Plasmodium-contaminated blood in the mosquito gut, we designed the concept of a Fe2+ and Lactate presence AND gate (figure 1).
Figure 1 - Rationale behind our AND gate.
Our approach was inspired by two previous iGEM projects:
Microbeacon from 2015 ETH_Zurich Team
Iron Coli from 2013 Evry Team
The team from Evry developed an interesting iron biosensor based on E. coli fur regulator, a protein that represses its cognate promoters in the presence of iron. Our chassis presents motifs for recognition of fur proteins, which can be explained by it's genetic similarity to E. coli. In order to avoid cross-talk between endogenous gene regulation and signal from our biosensor, we developed a new system for Fe2+ detection based on dtxR regulator,
Figure 2 - We designed a lactate and iron (Fe2+) AND gate as a way of detecting the contaminated blood. (A) Our biosensor device composed of generators of DtxR (BBa_K2486004) and LldR (BBa_K2486007), a PdtxR inverter based on tetR () and a reporter module with a hybrid promoter regulated by TetR and LldR ().
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).
