Part:BBa_K5378007
feaR-A81L
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
FeaR is an AraC family regulator that activates transcription of the tynA and feaB genes in Escherichia coli.FeaR-A81L was identified as PEA-specific variantss, with 580-fold induction.In our project, FeaR-A81L binds to phenylacetaldehyde (PAG) and induces the expression from PtynA.
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.
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).
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.
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