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LacI Inducible Module

Overview

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.

Usage

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 v0.1.0-001 Distribution Plate at 500 nM.

DNA Parts

pT7-lacI — 🧬Nucleus v0.1.0-001 Distribution Plate well G2.
pT7-lacO-plamGFP — 🧬Nucleus v0.1.0-001 Distribution Plate well G4.

Protein Components

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

Reaction Construction

Component	Reaction Volume (ul)
Master Mix
PURExpress Solution A	4
PURExpress Solution B	3
RNase I	0.5
pT7-lacO-plamGFP (10 nM)	0.5
lacI (10 mM)	0.5
Total	9

Per reaction
Master Mix	9
Inducer	1
Total	10

Performance Data

In vitro repression

Repression kinetics of pT7-lacO-plamGFP by LacI. Purified lacI protein was included in PURE reactions containing 5 nM plasmid DNA.

Repression of pT7-lacO-plamGFP by LacI at steady state. Purified LacI protein was included in PURE reactions containing 10 nM plasmid DNA.

Titration of purified LacI repressor protein shows repression across the range of 15.63—1000 nM LacI. 1000 nM LacI provides strong repression, allowing for suitable dynamic range when induced.

There is a small increase in expression at repressor levels around 62.5 nM. While this may be an artifact, we see a similar effect in some tet repressor data (🟤TetR Inducible Module) suggesting that it may represent a genuine behavior of the system. One possible mechanism is that increased repression of mRNA production in the reaction, to a point, preserves PURE’s productive capacity for translation of mRNA into protein.

The 250 nM repressor datapoint is likely a technical failure within that reaction.

Induction

Induction kinetics of pT7-lacO-plamGFP when induced with IPTG. Samples were repressed with 500 nM purified LacI protein. Positive represents a reaction without LacI repressor.

Steady state expression of pT7-lacO-plamGFP when induced with IPTG. Samples were repressed with 500 nM purified LacI protein. Positive represents a reaction without LacI repressor.

We are able to induce a repressed lacI reaction across a range of 500—2000 nM IPTG. These concentrations accord with those used to induce the system in living cellular systems (for instance, during protein expression in E. coli BL21(DE3) cells), as well as in cell-free lysates.

All induced samples outperform the unrepressed positive control sample (+). As in the repressor data, this may reflect an underlying biological behavior, or may have been caused by minor variances between the sample preparations. We do see this behavior replicate across different experiments, and are investigating it further.