Part:BBa_K5378023

PtynA-RBS-GFP

In our design,the promoter PtynA is activated, starting the expression of metabolic modules downstream.Then we designed a plasmid that can be transformed into EcN to express GS.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

In humans and other mammals, ammonia is majorly digested by the Urea Cycle in the liver. However, as it is shown before, the liver functions are destroyed in HE patients, so the urea cycle is hard to use. So we move our sight to plants. Plants utilize ammonium (NH₄⁺) as a nitrogen source through several processes, which are critical for their growth and development. Upon uptake by plant roots, ammonium is either directly assimilated into amino acids via the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway or converted to nitrate for further use in metabolic processes. Ammonium is an energetically favorable form of nitrogen compared to nitrate(like the annamox bateria), as it does not require reduction before assimilation. However, excessive ammonium can be toxic to plants, so its uptake and assimilation are tightly regulated to maintain optimal nitrogen levels within the plant tissues. As for the nitrate path, it may produce harmful side products such as nitrite, so we finally decided to mimic the GS-GOGAT cycle to metabolize ammonia in HE patients. So we expressed the glutamate synthase in EcN, and it can degrade ammonia into glutamine without harm to the body.

Figure 1.Metabolic Module

Functional Verification

In our design,the promoter PtynA is activated, starting the expression of metabolic modules downstream.Then we designed a plasmid that can be transformed into EcN to express GS.From the figure below, the size of each band of k y agarose gel electrophoresis is basically the same as the size of the target gene, indicating that the plasmid has been successfully transformed into ECN.

Figure 2.Transfer to bacterial pcr results with GS plasmids

Ammonia degrading ability of GS enzyme

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 3a). 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.

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 3b), and this trend remains when we extended coculturing time to 24 hours (Figure 3c). These results indicate the successful expression and function of the metabolic module.

Figure 3. 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). 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.

Altogether: Sensing-Metabolic System Validation

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 4a). Results showed that with the concentration of 50μM NH4Cl, 100ng/ml PEA induced the most significant decrease in ammonia (Figure 4b), which was consistent with the trend in both sensing and metabolic modules.

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 4c), 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.

Figure 4.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.

Reference

[1]Qvartskhava, N. et al. Hyperammonemia in gene-targeted mice lacking functional hepatic glutamine synthetase. Proc. Natl Acad. Sci. USA 112, 5521–5526 (2015).

[2]Frieg, B., Gorg, B., Gohlke, H. & Haussinger, D. Glutamine synthetase as a central element in hepatic glutamine and ammonia metabolism: novel aspects. Biol. Chem. 402, 1063–1072 (2021).

[3]Paluschinski, M. et al. Characterization of the scavenger cell proteome in mouse and rat liver. Biol. Chem. 402, 1073–1085 (2021).

[4] Haussinger, D. Nitrogen metabolism in liver: structural and functional organization and physiological relevance. Biochem. J. 267, 281–290 (1990).

[5]Gallego-Durán, R., Hadjihambi, A., Ampuero, J. et al. Ammonia-induced stress response in liver disease progression and hepatic encephalopathy. Nat Rev Gastroenterol Hepatol (2024). https://doi.org/10.1038/s41575-024-00970-9

[6]Hao DL, Zhou JY, Yang SY, Qi W, Yang KJ, Su YH. Function and Regulation of Ammonium Transporters in Plants. Int J Mol Sci. 2020 May 18;21(10):3557. doi: 10.3390/ijms21103557. PMID: 32443561; PMCID: PMC7279009.