PROOF OF CONCEPT

Our concept is based on three major devices, that can be tested and optimised seperatly, and can then, once they fulfill all of our strict criterions, be brought together. To prove our concept is working, we here present the three devices we decided to suit our needs best.

AND GATE

This part takes nitric oxyde (NO) and N-Acyl homoserine lactone (AHL) as it's inputs and activates downstream gene expression. We were able to construct multiple versions of this AND gate, tested each of them, and decided on how to optimize it to fit our purpose most: detect the physiological concentration ranges of NO and AHL during diseased as well as healthy state. Plot AND gate: Activation of the AND gate leads to expression of a fluorescent reporter (sfGFP, Part:BBa_K....). E. coli cells were induced with ... We can show that our AND gate has a X-fold activation compared to the negative control. This activation is assumed to be sufficient to activate its downstream device: The switch.

SWITCH

Our switch consists of a recombinase, that once expressed, irreversibly switches the reporter construct. Different recombinases were constructed: TP901, Bxb1, phiC31. Here again, we focus on optimisation before assembly of all the parts. We tested the recombinases codon optimized, non-optimized, with different RBS' as well as different degradation tags. Our favourite recombinase (codon optimized bxb1, Part: BBa_K...) exhibits the most desired kinetic data. Plot We conclude, that this switching rate is most desired for the whole construct.

REPORTER

Our reporter consists of an AHL inducible promoter flanked by recombinase specific att-sites. While not flipped, the reporter will express red fluorescent protein (mNectarine, Part:BBa_K..) once induced. If flipping occurs, the promoter will face the other direction and is able to express green fluorescent protein (sfGFP, Part:BBa_K..). Different reporter variants were tested by changing degredation tags. We found .... most suitable for our purpose. Plot:

CONCLUSION

All three BioBrick devices were tested individually and show the desired function. The system was modeled, and shown to be functional. We conclude, that the concept works. The system will be put together, and its function will be demonstrated under real-world conditions.

REFERENCES

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