Gene Regulation, Lac Operon / Lac Repressor

Introduction

3D structure of the lac operon binding to the lac repressor protein, showing the DNA operator, head group, N-terminal core, C-terminal core, binding pocket, and 4-helix bundle. — Figure 1. Lac operon binding to Lac repressor protein.

The regulation of the lac operon (lacO), a set of Escherichia coli genes responsible for lactose metabolism in the bacteria, by the lac repressor is a well-studied system in molecular biology. The lac repressor, a DNA-binding protein (lacI), is key to controlling the lac operon. The equilibrium dissociation constant (K_D) of this system is estimated to be in the nM range.

This lacO / lacI system is often introduced in the context of gene regulation in biochemistry and biology classes at the undergraduate level. More advanced research on lacO / lacI includes applications in imaging and chromatin editing.

In a broader context, SPR studies of protein–DNA complexes are important in understanding gene expression regulation pathways.

The binding strength (affinity, K_D) of the lacO / lacI system was determined with the P4SPR in an academic lab at the Université de Montréal. The easy-to-use P4SPR and user-friendly interface allowed lab technicians to develop an SPR assay to demonstrate this biological system in undergraduate lab classes.

Experiment Setup

1System Setup

System setup diagram showing P4SPR instrument connected to a computer and Au chips, alongside a lab photo of the benchtop P4SPR setup with laptop, instrument, solution vials, and injection syringes. — Figure 2. Benchtop P4SPR set-up.

The USB-powered P4SPR can be connected to a laptop to initiate operation control and data recording using the P4SPR Control software. With its user-friendly design, the P4SPR can be set up easily. The microfluidic cell (Figure 3) is capped over the gold sensor chip and tightly secured in the sensor chip cavity before injecting solutions in both sample and reference channels for baseline stabilization.

The S-shaped sample channel has 3 parallel sensing areas which can provide triplicate sample measurements along with one control measurement with the reference channel. Bubbles formed in the channels can be visually seen through the exposed clear channels for immediate removal to prevent any loss of signal.

Diagram of the P4SPR microfluidic cell showing four channel patterns (A, B, C, D) with their corresponding light interrogation positions. Channel A is the reference; channels B, C, D are sample channels. Inlet/outlet holes and flow direction are indicated. — Figure 3. P4SPR microfluidic cell. Channels B, C, and D provide triplicate sample measurements; channel A serves as the reference control.

2Experimental Procedures

System setup
(5 min)

Baseline stabilization

→

Surface preparation
(1 h)

Probe immobilization
MCH spacer immobilization
DNA hybridization
Wash

→

LacI solutions injection
(10 min each)

Signal stabilization
Inject lacI (5 nM)
Wash

→

Regeneration & repeat

Regeneration
Wash
Repeat with 20, 50, 100, 200 nM

Figure 4. Experimental workflow.

After baseline stabilization, thiolated lacO DNA was injected into the sample channel. Immobilization occurred spontaneously via strong gold–sulfur bond formation until surface saturation, shown by the SPR signal plateau. The complementary lacO oligomer was then injected to allow for hybridization. The sensor surface was washed thoroughly before the binding assay.

A stock lacI protein solution was used to prepare solutions of 5, 20, 50, 100, and 200 nM. Each solution of increasing concentration was sequentially injected and incubated for 10 minutes, with abundant washing solution and regeneration buffer in between to remove remaining lacI proteins from the previous injection.

Data from all 4 channels were recorded and then exported to Excel for further data analysis.

Results and Discussion

In this experiment, 3 parallel binding curves were generated from the sample channels. The signals were averaged and subtracted from the reference channel signal. The resulting SPR signal is plotted against time (Figure 5).

SPR signal in nm plotted against time in seconds, showing five sequential injection cycles for lacI at 5, 20, 50, 100, and 200 nM. Each cycle shows a binding association phase followed by a wash/dissociation phase. Signal magnitude increases with concentration. — Figure 5. SPR signal with respect to different concentrations of lacI over time.

The binding shifts corresponding to different concentrations of lacI are used to establish the binding isotherm and hence determine the K_D (Figure 6).

Affinity determination of LacO / LacI with P4SPR. Superimposed binding curves showing SPR shift in nm over time in minutes for lacI at 5, 20, 50, 100, and 200 nM. Higher concentrations reach larger signal plateaus and equilibrate faster, consistent with a KD in the low nM range. — Figure 6. Superimposed binding curves of increasing lacI concentrations.

K_D = 6.4 ± 1.2 nM

The measured K_D is in concordance with the literature-reported value in the same low nM range for lacI-based transcription repressor proteins.¹ Unlike the DNA binding assay used in the published paper, this SPR assay required no DNA radiolabeling and monitored binding interactions in real time.

The P4SPR Advantages

Modular & Mobile

Compact, USB-powered design allows benchtop or field deployment with minimal infrastructure.

Multichannel

Four simultaneous channels (3 sample + 1 reference) enable triplicate measurements with built-in reference subtraction.

Minimal Sample Prep

No purification kits or radiolabels required — rapid setup saves time and reduces reagent costs.

High Sensitivity

Thin-film SPR provides deep evanescent field penetration for accurate affinity determination in the nM range and beyond.

Conclusion

The P4SPR successfully characterized the binding interaction of the lac operon DNA and its repressor protein, and the result was validated with published literature. This experiment allowed undergraduate students to familiarize themselves with the technology and its application in monitoring biomolecular interactions.

The P4SPR is also a useful tool for characterizing other biological systems such as DNA–DNA, RNA, and aptamer pairs, helping scientists better understand binding and uncover function in biological pathways.

Acknowledgements

We would like to thank Philipe Lampron, Shona Teijeiro, and Sébastien Truche of the Université de Montréal Biochemistry department for developing the assay.

¹ Tungtur et al. Reconciling in vitro and in vivo activities of engineered, LacI₃-based repressor proteins: Contributions of DNA looping and 4 operator sequence variation. doi: 10.1101/477893