Team:Paris Bettencourt/Proteins Caging

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@iGEM_Paris

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.

Dronpa is one of the reversible photoswitchable fluorescent proteins (RSFPs), these are proteins that are switched on and off reversibly by specific wavelengths. Dronpa is switched on by default “fluorescent” and is switched off when illuminated by cyan light (~500nm). Dronpa Fluorescence is recovered by shining violet light (~400nm)

Figure 1: an illustration of the on/off switching of dronpa and the associated alternation between the monomer/dimer structures

The conformational changes that are associated with the on/off switching of Dronpa Lys145Asn have been used in a design that facilitates the optical control of protein activities. When Dronpa domains are fused to both termini of an enzyme of interest, the Dronpa domains form a tetramer and cage the enzyme leading to its inactivation. By Shining cyan light, Dronpa is switched off and the tetramer dissociates into monomers, as a result, the caged enzyme is activated (1) (3).

Figure 2: A fluorescent light-inducible protein design based on Dronpa Lys145Asn- From Zhou, X.X. and Lin, M.Z., 2013.

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:

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
bettencourt.igem2017@gmail.com