Part:BBa_K5378006

tynA-G494S

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 267
  Illegal AgeI site found at 522
  Illegal AgeI site found at 2074
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

TynA is a periplasmic amine oxidase containing copper and topaquinone cofactors, which converts amines to aldehydes.TynA-G494S discovered by Rottinghaus, A. G. et al converts phenethylamine (PEA) to phenylacetaldehyde (PAG) specifically.

Figure 1.TynA

Functional Verification

We designed a plasmid that can be transformed into EcN to express GFP,which is used to determine whether or not the sensing module will operate effectively.From the figure below, the size of each band of 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.PtynA-RBS-GFP

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 2a).

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 2b), 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 2c).

Figure 3.Validation of the feasibility of the sensing module.(a)Schematic representation of the construction and mechanism of engineered EcN with sensing module. EcN was co-transformed with plasmid Pcon-FeaR-Pcon-TynA and plasmid PTynA-GFP via electroporation. After co-culturing with different concentration of PEA for different time, fluorescence intensity was measure by microplate reader and fluorescence microscopy.(b)Fluorescence Intensity with 100ng/ml, 50ng/ml, 25ng/ml, 5ng/mland 0ng/ml PEA cocultured in engineered EcN. The fluorescence was measured on microplate reader by excitation at 410 nm and detection of emission at 500 nm. OD600 (absorbance of 600nm) was also measured on microplate reader for normalization. Data shows mean±SD, n=3 independent experiments.(c)Fluorescence observation of the Pcon-FeaR-Pcon-TynA and PTynA-GFP engineered bacteria fluid cocultured with different concentrations of PEA. Fluorescence was observed after 12-hour co-culturing through fluorescence microscopy.

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.