From 2012.igem.org

Overall project

Drafted crystal structuce of the LOV-domain. The Jα-helix is shown in blue, the flavin mononucleotide and the functional cysteine residue are depicted as balls and sticks.

Control of protein activity at the peptide level offers spatial-temporal control and quick reaction times, but so far has always involved target-specific tools, such as specific chemical inhibitors or proteases. We are developing and characterizing a fusion construct containing a light sensitive domain that provides quick, universal peptide-level light control of proteins of interest within the framework of easy biobrick-conform coupling.

We are engineering the LOV (Light, Oxygen, Voltage) domain – commonly found in plants where it enables light-directed growth – to control protein activity through blue light. Upon exposure, the LOV domain undergoes a conformational change and shifts away from the protein of interest, uncaging the coupled protein and allowing it to resume activity. In our project, we coupled a small part of the beta-galactosidase (which offers a simple assay) to the LOV domain as a proof-of-principle. We also built a cell death device using ccdB, a gyrase inhibitor. Furthermore we will design a MazF construct, as an example for a Hydrolase.

Potential applications of our LOV fusion system include bioreactor regulation or site-specific drug activation.

Project Details

LOV-Blues

In our proof-of-principle, we are coupling LacZalpha to the LOV domain. LacZalpha is one of two parts of a split-version beta-galactosidase, which upon exposure to light will resume galactosidase activity in mutants containing LacZomega, the complimentary second part of beta-galactosidase. Using a chromophore substrate for our beta-galactosidase gives us a simple blue-to-white assay.

LOV Kills / LOV-Ccdb

To show one of the many, wide-ranging possible applications, we will fuse a cell death protein, ccdB, to our LOV construct. Upon light exposure, the cell will go into apoptosis.

LOV Cuts / LOV-MazF

MazF is a ACA-specific ribonuclease from B. subtilis which in nature is used for toxin/antitoxin addiction systems. It leads to programmed cell death in bacteria upon high-scale expression, thus qualifying it for use in cell death devices.

Fusion System

Our Approach

For detailed information about the experiments we did in the lab visit our Notebook.