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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.

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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