Team:Evry Paris-Saclay/Proof Of Concept
**Figure 1.** Lysate based cell-free systems principle and advantages (adapted from [[3]](#ref3)).
## Design
In this new platform, for time constraints reasons, we tested the first-generation of Rosewood toehold switches and triggers the design of which is described on the ["Model"](https://2020.igem.org/Team:Evry_Paris-Saclay/Model) page of this wiki. After the *in vivo* screening described in the ["Engineering Success"](https://2020.igem.org/Team:Evry_Paris-Saclay/Engineering) page of this wiki, the best candidates were selected to be tested in the cell-free final assay (Table 1).
**Table 1.** Rosewood switches and cognate triggers.
| Label | sfGFP expression cassettes’ part numbers | Trigger transcriptional units’ part numbers |
|---|---|---|
| Rosewood DmMatK Toehold Switch 1.1 | BBa_K3453111 | BBa_K3453121 |
| Rosewood DmMatK Toehold Switch 1.3 | BBa_K3453112 | BBa_K3453122 |
| Rosewood DmMatK Toehold Switch 1.4 | BBa_K3453113 | BBa_K3453123 |
| Rosewood DmRbcL Toehold Switch 1.1 | BBa_K3453131 | BBa_K3453141 |
| Rosewood DmRbcL Toehold Switch 1.3 | BBa_K3453133 | BBa_K3453143 |
| Rosewood DmTrnL-UAA Toehold Switch 1.3 | BBa_K3453153 | BBa_K3453163 |
**Figure 2.** Characterization of sfGFP expression controlled by the Rosewood toehold switches and triggers in an *E. coli* BL21 Star™(DE3) based cell-free system. The data and error bars are the mean and standard deviation of at least three measurements.
**Figure 3.** Fluorescence fold change of the six Rosewood toehold switches tested in an *E. coli* BL21 Star™(DE3) based cell-free system.
## Conclusion
We have characterized the function of 6 of our own designed Rosewood toehold switches in an *E. coli* based cell-free system. The testing conditions were set up to be as close as possible to the real conditions in which our Rosewood detection tool will be implemented: a cell-free system supplemented with trigger Rosewood RNA.
Here we demonstrate the efficiency of 5 Rosewood toehold switches as a readable output was produced only in the presence of Rosewood RNA. Notably, we obtained at least one functional cell-free sensor for each of the three *Dalbergia maritima* genes tested: MatK, RbcL and TrnL-UAA.
Thus we bring a Proof of Concept that gives a green light for the future improvements described in the ["Implementation"](https://2020.igem.org/Team:Evry_Paris-Saclay/Implementation) page of this wiki.
## References
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