In the studies so far, biosensor studies have been conducted to combine one type of reporter gene with PyeaR promoter (BBa_K216005) to see the expression according to the nitrate concentration. However, in this project, our team used two reporter genes (GFP, RFP) along with the PyeaR promoter to allow the usage as an Arduino sensor. We also used the lon protease system to express different colors (Green → Yellow → Red) according to the nitrate concentration. More specifically, our nitrate biosensor is designed to express the color red under conditions in which nitrate exists, and green under normal conditions.
Therefore, the red color was designed to be controlled by the yeaR promoter, which enables the expression of mScarlet, as mentioned above. The green color was designed to be controlled by the amount of mScarlet produced, other polycistronic-arranged repressors (cI, MerR, EilR, CymR), and lon protease. Under normal conditions, NsrR, yeaR promoters are inactivated, preventing the expression of the repressor and Ion protease. As a result, mVenus is expressed and maintained. In our experiment, by introducing the previously known negative inducible expression system pJEx-D/EilR (Ruegg et al., 2018), pCuO/CymR (Seo & Schmidt-Dannert, 2019), pL/cI (Valdez-Cruz et al., 2010) and pHTH/MerR, we controlled the expression of mVenus by using the negatively regulated promoter (pJEx-D, pCuO, pL, pHTH), making a green fluorescent protein to form under normal conditions that repressor is absent. Yet, when exposed to nitrogen, the yeaR promoter is activated to form the repressor and Lon protease along with mScarlet, and the expression of mVenus is suppressed by the repressor. The mVenus, which is fused with the lva tag at the c-terminal, quickly decomposes by the lon protease (Cameron & Collins, 2014), causing the green color to fade away and red color to appear.
Using these principles, our nitrate biosensor can easily measure the concentration of nitrate, which causes eutrophication, with the chromatic colors green and red. We created beads that contain our modified E. coli, and it can show different colors depending on the nitrate concentration by measuring the amount of fluorescence expressed using UV lights on the beads. Since the color can be changed into electrical signals using the Arduino color sensor, it is possible to obtain a nonexisting nitrate sensor. Furthermore, as the nitrate concentration is measured by measuring the intensity of the two types of fluorescence, more accurate measurement of nitrate concentration is possible.
References
- Ruegg, T. L., Pereira, J. H., Chen, J. C., DeGiovanni, A., Novichkov, P., Mutalik, V. K., Tomaleri, G. P., Singer, S. W., Hillson, N. J., & Simmons, B. A. (2018). Jungle Express is a versatile repressor system for tight transcriptional control. Nature communications,9(1), 3617.
- Seo, S.-O., & Schmidt-Dannert, C. (2019). Development of a synthetic cumate-inducible gene expression system for Bacillus. Applied microbiology and biotechnology, 103, 303-313.
- Valdez-Cruz, N. A., Caspeta, L., Pérez, N. O., Ramírez, O. T., & Trujillo-Roldán, M. A. (2010). Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters. Microbial cell factories, 9(1), 18. https://doi.org/10.1186/1475-2859-9-18
- Cameron, D. E., & Collins, J. J. (2014). Tunable protein degradation in bacteria. Nature biotechnology, 32(12), 1276-1281.