Team:Paris Bettencourt/Proteins Caging
PROTEIN PHOTOCAGING
Introduction
Photoreceptors are valuable optogenetic tools which, upon coupling with other proteins, activate certain functions in a controlled spatial and temporal manner when exposed to the appropriate wavelength of light. However, the usage of photoreceptors suffers from many drawbacks including the toxicity of the light to the cells, photobleaching of the receptors and the delay in the response i.e. the time needed for transcription and translation of the target protein to be controlled-. The emergence of Fluorescent light-inducible proteins is an attractive alternative that doesn’t suffer from these drawbacks.
Reversible photoswitchable fluorescent proteins (RSFPs) are proteins that are switched on and off reversibly by specific wavelengths. a K145N mutant of the fluorescent protein Dronpa -which is a monomeric Reversible photoswitchable protein derived from a tetrameric parent has been used to control protein activity. When two copies of Dronpa are added to proteases or kinases the 2 copies dimerize and cage the protein leading to its inactivation. By shining a 500 nm cyan light, dronpa is switched off, turns into monomers and as a result the caged enzyme is activated
Design of protein Caging
In our design, we had two copies of Dronpa Fluorescent Protein with two BsaI cutting site in between to allow the insertion of our genes of interest.
Caging several repressors using Dronpa
Repressors bind DNA and setback transcription. In our project we developed a logic gate at the promoter level by creating dually repressed promoters using different combinations of the operators for TetR, P22 c2 and HK CI and it was interesting to us to test if these repressors can be controlled by light. TetR, HK CI and P22 C2 function as homodimers. Our original hypothesis was that caging with Dronpa will prevent them from dimerization. To test this hypothesis we created the following constructs:
