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. Source: RCSB Protein Data Bank

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

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

Fusion System

The Experiments

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