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TetR Inducible Module

Overview

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 anhydrotetracycline (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. Anhydrotetracycline is membrane-permeable which means that the alpha-hemolysin membrane pore is not required for induction, although it can be used.

Usage

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 Distribution Plate.

DNA Parts

pT7-tetR — 🧬Nucleus v0.1.0 Distribution Plate well G1.
pT7-tetO-plamGFP — 🧬Nucleus v0.1.0 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

Performance Data

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 kinetics of pT7-tetO-plamGFP by tetR.

Repression of pT7-tetO-plamGFP by tetR at steady state.

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 kinetics of pT7-tetO-plamGFP by tetR. tetR repressor protein is present at 500 nM. Positive control is pT7-tetO-plamGFP without TetR repressor protein.

Induction of pT7-tetO-plamGFP by tetR at steady state. tetR repressor protein is present at 500 nM. Positive control is pT7-tetO-plamGFP without TetR repressor protein.

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

tetR detector synthetic cell induced at multiple anhydrotetracyline concentrations. 8 timepoints are displayed per sample condition, with approximately 22 minutes per timepoint. The detector cells were incubated for a total of 12 hours. First row: induction of tetR detectors using 625 nM, 312.5 nM, and 0 nM (fully repressed) anhydrotetracycline, introduced into the outer buffer. Second row: induction of tetR detector with 2500 nM anhydrocycline in the inner solution, and positive control without tetR repression.

GFP expression within synthetic cells when induced with 312.5 uM anhydrotetracycline.

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.

Resources and References

Credits