Part:BBa_K5378005

feaR-A81L-tynA-G494S-PtynA-TPH1

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1179
  Illegal AgeI site found at 1434
  Illegal AgeI site found at 2986
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

In order to better identify PEA, we introduce the FeaR-A81L and TynA-G494S mutant with 580-fold induction and PEA specificity.The variant induces a 580-fold enhancement in expression and is PEA-specific.

Functional Verification

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 1a). Results showed that with the concentration of 50μM NH4Cl, 100ng/ml PEA induced the most significant decrease in ammonia (Figure 1b), 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 1c), 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 1.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. CAPTION_HERE

Reference

[1] Rottinghaus, A. G., Xi, C., Amrofell, M. B., Yi, H., & Moon, T. S. (2022). Engineering ligand-specific biosensors for aromatic amino acids and neurochemicals. Cell systems, 13(3), 204-214.

[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.