<p>To demonstrate that PEA, a reliable risk factor of HE identified by the current work of our secondary PI (see details in our Design page) , could initiate the downstream gene circuit, we first engineered Escherichia coli Nissle 1917(EcN) to produce FeaR and TynA constantly by transforming EcN with plasmid Pcon-tynA-Pcon-feaR. Thereby, PEA could be degraded by the enzyme TynA into PAG and PAG could bind with FeaR as a transcriptional factor, which could activate the inducible promoter PTynA. Then we transformed the engineered EcN with plasmid PTynA-GFP to demonstrate the feasibility and efficiency of sensing module via fluorensence (Figure 1a).</p>
<p>To demonstrate that PEA, a reliable risk factor of HE identified by the current work of our secondary PI (see details in our <ahref='https://2024.igem.wiki/smu-gdmu-china/design'>Design page</a>) , could initiate the downstream gene circuit, we first engineered Escherichia coli Nissle 1917(EcN) to produce FeaR and TynA constantly by transforming EcN with plasmid Pcon-tynA-Pcon-feaR. Thereby, PEA could be degraded by the enzyme TynA into PAG and PAG could bind with FeaR as a transcriptional factor, which could activate the inducible promoter PTynA. Then we transformed the engineered EcN with plasmid PTynA-GFP to demonstrate the feasibility and efficiency of sensing module via fluorensence (Figure 1a).</p>
<p>After coculturing with 0, 5, 25, 50 and 100ng/ml PEA for 12 hours, results showed a significant increase in fluorensence under microscopy, along with the the increased level in PEA concentration (Figure 1b), suggesting a successful expression and high feasibility of the sensing module. Moreover, the fluorescent intensity under different concentrations of PEA throughout 24 hours also verified that our engineered EcN could indeed be more sensitive to the increase in PEA concentration (Figure 1c).</p>
<p>Our metabolic module aims to degrade the over-accumulated NH3 in patients' gut by expressing GS, an enzyme that could utilize NH4+ and metabolize it into glutamine which does no harm to the human body. To validate the feasibility of this module, we transformed EcN with plasmid Ptac-GS and used IPTG to induce the expression of GS (Figure 2a). Meanwhile, we transformed EcN with the vector plasmid PET-32a as control gruop and coculture them with differnt concentratioon of NH4Cl in M9 minimal culture medium (Why in M9 rather than the regular LB medium? See details in our Engineering Page).</p>
<p>Our metabolic module aims to degrade the over-accumulated NH3 in patients' gut by expressing GS, an enzyme that could utilize NH4+ and metabolize it into glutamine which does no harm to the human body. To validate the feasibility of this module, we transformed EcN with plasmid Ptac-GS and used IPTG to induce the expression of GS (Figure 2a). Meanwhile, we transformed EcN with the vector plasmid PET-32a as control gruop and coculture them with differnt concentratioon of NH4Cl in M9 minimal culture medium (Why in M9 rather than the regular LB medium? See details in our <ahref='https://2024.igem.wiki/smu-gdmu-china/engineering'>Engineering Page</a>).</p>
<p>To our joy, the ammonia level in EcN_GS group cocultured with 50μM NH4Cl for 12 hours is significantly lower than the control group (Figure 2b), and this trend remains when we extended coculturing time to 24 hours (Figure 2c). These results indicate the successful expression and function of the metabolic module.</p>
<figcaptionclassName='caption'>Figure 2. Validation of the feasibility of the metabolic module. (a)Schematic representation of the construction and mechanism of engineered EcN with metabolic module. EcN was transformed with plasmid Ptac-GS via electroporation. After co-culturing with different concentration of NH4Cl for different time, NH3 concentration was measured and calculated by NH3 detection kit based on indigol blue reaction via microplate reader (OD 630nm). The structure of GS was predicted based on AlphaFold3. (b)NH3 concentration after coculturing 0μM, 0.5μM, 5μM and 50μM NH4Cl with engineered EcNs for 12 hours. EcN_vector was transformed with the vector plasmid, pET-32a. Data shows mean±SD, n=3 independent experiments. (c)NH3 concentration in 0h, 4h, 8h, 12h and 24h after coculturing 50μM NH3Cl with engineered EcNs. Data shows mean±SD, n=3 independent experiments.</figcaption>
<h3>Tackling Intrinsic Ammonia of Engineered EcN</h3>
<p>Interestingly, the level of NH3 increased rather than decreased as we expected throughout 24 hours after coculturing with additional NH4Cl. This can be explained by the complicated metabolic curcuits of nitrogen, especially the urea metabolism[1]. Therefore, we tried to seek paths to knock out relative genes to lower the intrinsic ammonia production (See details in our Model Page) but due to time limit, we didn't conduct wet lab experiments, which is well-planned in our future optimization.</p>
<p>Interestingly, the level of NH3 increased rather than decreased as we expected throughout 24 hours after coculturing with additional NH4Cl. This can be explained by the complicated metabolic curcuits of nitrogen, especially the urea metabolism[1]. Therefore, we tried to seek paths to knock out relative genes to lower the intrinsic ammonia production (See details in our <ahref='https://2024.igem.wiki/smu-gdmu-china/model'>Model Page</a>) but due to time limit, we didn't conduct wet lab experiments, which is well-planned in our future optimization.</p>
<h3>Safety concerns of GS enzyme to degrade normal level of ammonia</h3>
<p>According to a review in Journal of Hepatology[2] , the physiological level of blood ammonia is around 35~50μM, a level of 120μM is associated with high risk of death in HE patients.</p>
<p>As shown in Figure 2b, the difference between EcN_vector and EcN_GS with lower NH4Cl concentration such as 5μM decreased and was even not significant with 0.5μM. This indicates that the GS enzyme requires a rather high level NH4Cl to reach the most efficient status. Therefore, when the level of ammonia is in a normal range, the metabolic module is not likely to cause a significant decrease in ammonia, which might be useful in other metabolic cycles. </p>
