Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Nikola Dyulgyarov
Date created: 2015-09-16 11:00:00
Date modified: 2015-09-17 09:32:14
PLTet0-1:NarL, PdcuS:sfGFP, J23109:TetR
| Types | DnaRegion |
| Roles | engineered_region Device |
| Sequences | BBa_K1682018_sequence (Version 1) |
Description
Functional nitrate anion sensor based on the NarX/NarL Two-Component Signal Transduction System in E. coli with semi-tunable dynamic range and sensitivity. NarL expression is regulated by the TetR-PLTet0-1 1-component regulatory system, whereby addition of ATC (anhydrotetracycline) induces higher NarL expression. This regulates the ratio of native NarX/NarL protein in the cell, controlling to an extent the dynamic range and sensitivities of the sensor.Phosphorylated NarL binds to the repressible PdcuS promoter from E. coli, regulating the expression of sfGFP.
Low nitrate conditions result in high sfGFP expression, high nitrate conditions repress transcription.
The sensitivity of the sensor is in the sub-mM to mM range for nitrate.
Further testing is needed to fully characterize the relationship of ATC-induction to nitrate sensitivity and dynamic range. The sensor is functional in the absence of ATC with a low dynamic range, and ATC concentrations of 1-10 ng/ml increase dynamic range and sensitivity. Further increase of ATC drives the ON-state down, decreasing dynamic range.
Notes
The PdcuS promoter was chosen for its NarL binding site, repressible activity, and the absence of other protein-binding domains, suggesting a robust, orthogonal response.The construct was assembled using Golden Gate Assembly.
Source
NarL, PdcuS - E. coli MG1655 genomesfGFP,TetR-PLTet0-1 scaffold - pRS334 (courtesy of Tabor Lab, Rice Univeristy)