lux pL controlled luxR with lux pR controlled gfp (LVA-tag)

Lux pL controlled LuxR + lux pR autoinducing LuxI (lva tag) + lux pR controlled GFP(lva tag).

The lux pL controlled luxR with lux pR is known as quorum sensing promoter. the lux pR is at slightly activated under normal circumstance and produces LuxI protein that synthesize a kind of acyl-homoserine lactone (AHL), which is a small molecule that can diffuse across the cell membrane and mediate intercellular coupling when it reaches the threshold as enough biomass accumulated. AHL will bind intracellular protein LuxR, which is also consecutively produced by luxR gene. The LuxR-AHL complex can activate the luxI promoter, and the positive feedback loop is built. That means once the biomass of bacteria reach the threshold, not only the expression of luxI protein gene will be increased but also that of the GFP downstream of the same quorum sensing promoter.

Usage and Biology

the GFP molecule acts as a photosensitizer, releasing free radicals upon exposure that produce reactive oxygen species (ROS) including H2O2. At the peak of oscillation, considerable vapour-phase H2O2 is produced by exposing GFP containing cells to fluorescent light. Conversely, at the trough of oscillation, cells contain almost no GFP, and therefore produce very little H2O2 upon fluorescing. Bursts of light thus generate bursts of H2O2 vapour whose concentration depends on the oscillating GFP level, just as periodic production of NDH-2 did previously. Indeed, this strategy was similarly able to synchronize our sensor array. Numerous controls were performed to ensure that synchronized oscillations did not occur at low fluorescence intensities.

Protocol

Construction

This part is constructed with quorum sensing promoter luxR, GFP with LVA tag, and LuxI. Via digestion and ligation, these parts are ligated as a composite.

Microfluid

SDS page

Experimental Data

SDS page, microfluid, and gel image of construction.

Sequence and Features

Assembly Compatibility: