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Team:Bonn/Project
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Overall project
Fusion protein design has always been time- and design-intensive, to say the least. We are developing and characterizing a fusion construct containing a light sensitive domain, providing easy coupling and light activation of proteins of interest to investigators, thus developing a protein-level light-induced knockout.
Using the LOV (Light, Oxygen, Voltage) domain commonly found in plants, where it enables light-directed growth, we are establishing guidelines for coupling proteins of interest to the LOV domain, which allows control of protein activity through blue wavelength light. Designing our reversible knockout at the protein level allows quick response times (2,2 microseconds activation time, 85 seconds deactivation time).
Project Details
What we intend to do
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. 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.
What you can do with it later
Possible uses for this device could encompass e.g. disinfection on a laboratory scale or mutant selection via blue light. Other possible applications of our LOV fusion protein include bioreactor control and regulation or site-specific therapeutic activation of antibody-coupled drugs in a patient's body.
The Experiments
What we did in the lab
For detailed information visit our Notebook.
