Fig. 1. Optimized nsrR expression system and nitrate induced reporter system
Our team's nitrate sensor (BBa_K4948031) was more sophisticated, based on the PyeaR promoter (BBa_K216005) from Edinburgh iGEM 2009. Since nsrR is a nitrate-dependent transcriptional repressor, a high amount of nsrR requires a high concentration of nitrate, resulting in a low performance sensor. Therefore, it is essential to determine the optimal concentration of nsrR. To this end, we used different constitutive expression units to control the intracellular concentration of nsrR. In addition, we added the hmpA1 site, which is known as the binding site of nsrR, between the yeaR promoter and the ribosome binding site.
Fig. 2. Measuring the strength of a promoter with the reporter system
Fig. 3. Changes in fluorescence intensity depending on the strength of the ribosome binding site in nitrate sensing
Our sensor is capable of detecting trace amounts of nitrate than existing sensors (Figure 4). To increase the intensity of nitrate-induced fluorescence, we added a strong RBS between the yeaR promoter and reporter gene, and in addition to this, we used a medium with higher nutrients to compensate for nitrate-induced cytotoxicity.
Fig. 4. Fluorescence intensity by nitrate concentration