To monitor nitrate in streams, we first constructed a biosensor using molecular biology and genetic engineering at the laboratory level. We wanted our biosensor to show green at low nitrate conditions and red at dangerous levels of concentration. For this purpose, we used a strong constitutive promoter (pJEx-D-RBS) to show green color under normal conditions (BBa_K4948033). However, in order to degrade the already made GFP when nitrate is present in high concentration, a lva tag that can be recognized by protease was fused to the gfp. And to control at the transcriptional level, EilR, a repressor that negatively regulates the transcriptional activity of pJEx-D, was added to the nitrate induced system.
At the laboratory level, we found that our biosensor worked smoothly enough to be distinguished by the eye in 6 hours.
The transformed DH5a containing the two plasmids that make up our nitrate sensor was encapsulated using Ca-alginate to enable real-time monitoring of nitrate in rivers and lakes in the real world. This process reduces the risk of leakage into the environment and sterilizes it before it is used for sensing, with UV treatment to measure fluorescence. The alginate encapsulated by our nitrate biosensor, Monitro, confirmed that the beads did indeed express mScarlet upon nitrate treatment.
To quantitatively show this result, we measured the fluorescence value using an RGB sensor and implemented it into an app so that users can easily and intuitively recognize it.
