<h2>Altogether: Sensing-Metabolic System Validation</h2>
<p>To demonstrate the function of our system after assembly, we co-transformed EcNs with plasmid Pcon-tynA-Pcon-feaR and plasmid PTynA-GS and coculture the engineered bacteria with different concentrations of PEA and NH4Cl (Figure 3a). Results showed that with the concentration of 50μM NH4Cl, 100ng/ml PEA induced the most significant decrease in ammonia (Figure 3b), which was consistent with the trend in both sensing and metabolic modules. </p>
<p>We also transformed plasmid Pcon-tynA-Pcon-feaR into EcN as the control group, and cocultured them with 100ng/ml PEA and 50μM NH4Cl for 4,8,12 and 24 hours. Results demonstrated a significant ammonia decrease in experiment group compared with the control group (Figure 3c), indicateing that a rahter high level of PEA could iniitate downstream metabolic module to express GS and resulted in the decrease of over-accumulated ammonia.</p>
<figcaptionclassName='caption'>Figure 3. Functionality verification of the PEA-sensing NH3-metabolizing system. (a)Schematic representation of the process of sensing and metabolic module. EcN was co-transformed with plasmid Pcon-FeaR-Pcon-TynA and plasmid PTynA-GS via electroporation. (b)NH3 concentration after coculturing different concentration of PEA and NH4Cl with engineered EcN for 12 hours. Data shows mean±SD, n=3 independent experiments. (c)NH3 concentration after coculturing 100ng/ml PEA and 50μM NH4Cl engineered EcN for 0, 4, 8,12 and 24 hours. EcN-FeaR-TynA was transformed with only plasmid Pcon-FeaR-Pcon-TynA as the control group. Data shows mean±SD, n=3 independent experiments.</figcaption>
