The TetR inducible expression module is a set of two genetic constructs that encode tetracycline inducible gene expression: pT7-tetR, encoding the repressor protein, and pT7-tetO-plamGFP, encoding an inducible T7 promoter.
The pT7-tetO-plamGFP construct constitutively expresses the open reporter plamGFP in the absence of repressor protein. The inducible promoter is also a MoClo Level 0 ‘P’ part, and may be assembled into a Level 1 transcription unit with other MoClo compatible genes.
Addition of Tet repressor (TetR) protein to the system, whether as a purified protein or via constitutive expression of the pT7-TetR construct, inhibits expression of the pT7-tetO-plamGFP construct. The mechanism of action is steric inhibition of the promoter site via Tet repressor binding to the tetO operator site.
Addition of anhydrotetracline (aTc) to the system leads to recovery of expression of the pT7-tetO-plamGFP construct. The mechanism of action is allosteric binding of anhydrotetracycline (aTc) to lac repressor causing weakened binding and release of Tet repressor from the tetO site.
The TetR module may be implemented by assembling the pT7-tetO-plamGFP inducible DNA construct into a standard PURE reaction, following 
Assemble PURE Reactions. Add purified TetR protein to a final concentration of 500 nM, or the pT7-tetR DNA construct from Nucleus v0.1.0-001 Distribution Plate.
DNA Parts
pT7-tetR— Nucleus v0.1.0-001 Distribution Plate well G1.pT7-tetO-plamGFP— Nucleus v0.1.0-001 Distribution Plate well G3.
Protein Components
- TetR purified protein—MedChemExpress HY-P71520, resuspended to 10 uM.
Reaction Construction
Component | Reaction Volume (ul) |
Master Mix | |
PURExpress Solution A | 4 |
PURExpress Solution B | 3 |
RNase I | 0.5 |
pT7-tetO-plamGFP (10 nM) | 0.5 |
tetR (10 mM) | 0.5 |
Total | 9 |
Per reaction | |
Master Mix | 9 |
Inducer | 1 |
Total | 10 |
The TetR module was validated in NEB PURExpress reactions as assembled using the Assemble PURE Reactions protocol. Purified repressor protein (MedChemExpress, HY-P71520) and anhydrotetracycline inducer (Cayman Chemical, 10009542) were added to the standard reaction to the final concentrations indicated. pT7-tetR-plamGFP plasmid DNA was added to a final concentration of 0.5 nM.
In vitro repression with tetR
Repression follows a roughly linear trend between 125 and 750 nM and saturates around 500 nM, though it can be further improved by increasing concentration to 2000 nM. We selected 500 nM as the target tetR concentration, optimizing for effective repression as well as efficiency in the amount of protein consumed. The steady state data also demonstrates a trend we noticed in the LacI Inducible Module, where a middling repression level appears to improve expression slightly.
In vitro induction of tetR
Induction with tetR using aTc is confounded by the yellow color of aTc, which overwhelms GFP fluorescence at high concentrations (greater than 50-100 uM). Such high concentrations also appear to negatively effect expression generally, potentially explained by tetracycline’s antibiotic action on the E. coli ribosome and the potential for aTc to have high-concentration antibiotic activity particularly in the absence of the protective tet-operon protein TetO.
An inducer concentration of 2.5-5 uM provides good induction and is well below saturating/toxic aTc levels. We again see an improvement in steady-state expression above an unrepressed positive control. Dynamic range under induction requires further optimization.
Encapsulated induction of tetR
The tetR detector cell appears to function when induced with low (312.5 uM) anhydrotetracycline. Higher aTc concentrations either begin to inhibit expression, or confound analysis by saturating the solution with background aTc fluorescence. Moreover, aTc at higher concentrations localizes to the liposome membrane, particularly during early timepoints, causing the membrane to be saturated with green fluorescence.
