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The lac inducible expression module is a set of two genetic constructs that encode IPTG inducible gene expression. pT7-lacI encodes the lac operon repressor protein, LacI. pT7-lacO-plamGFP is a GFP reporter construct, which is repressed by the LacI protein and may be induced with the sugar Isopropyl β- d-1-thiogalactopyranoside (IPTG).

The pT7-lacO-plamGFP construct constitutively expresses the plamGFP reporter in the absence of the repressor protein LacI. 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 LacI protein to the system, whether as a purified protein or via constitutive expression from the pT7-LacI construct, inhibits expression of the pT7-lacO-plamGFP construct. The mechanism of action is steric inhibition of the promoter site via lac repressor binding to the LacO operator site.

Addition of IPTG to the system leads to recovery of expression of the pT7-lacO-plamGFP construct. The mechanism of action is allosteric binding of IPTG to lac repressor causing weakened binding and release of lac repressor from the LacO site.

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The LacI module may be implemented by assembling the pT7-lacO-plamGFP inducible DNA construct into a standard PURE reaction, following Assemble PURE Reactions. Add purified LacI protein to a final concentration of 500-1000 nM, or the pT7-tetR DNA construct from Nucleus v0.1.0 Distribution Plate at 500 nM.

DNA Parts

• pT7-lacI — Nucleus v0.1.0 Distribution Plate well G2.
• pT7-lacO-plamGFP — Nucleus v0.1.0 Distribution Plate well G4.

Protein Components

• LacI purified protein—MedChemExpress HY-P70247, resuspended to 10 mM.

Reaction Construction