Optogen.net
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This is my personal web page describing some of the optogenetics projects that I have worked on in the past years. This is not the homepage of the "International Workshop on Technologies for Optogenetics and Neurophotonics", which you can find here: https://optogen.eu/

We utitilize plant light receptor domains to build light controllable versions of proteins that we are interested in. Such light controllable proteins can be turned off or on in split second, and allow us to control proteins at the subcellular level. One of the light receptor domains that we utilize is called a LOV2 domain. This domain is part of a plant protein named phototropin, which in plants is essential for growth towards sunlight(phototropism). The LOV domain is particularly sensitive to blue light.
Phototropism in action
Arrow indicates direction blue light
(mixed vegetable sprouts)

Here are links to our papers on optogenetic control of proteins in mammalian cells:

Below is a link to our paper on a blue light inactivated version of the microtubule plus-end-tracking protein EB1, which was published in Nature Cell Biology in 2018. EB1 is an important regulator of the microtubule cytoskeleton in eukaryotes. It can regulate MT growth dynamics and it can mediate attachement of MTs to other subcellular structures, by recruiting over 40 different types of +TIPs to growing MT ends. We engineered a variant of EB1 which splits apart in the presence of blue light, and can no longer recruit these other +TIPs to the ends of MTs. This impacts MT growth, and MT attachment at the cell cortex. We engineered pi-EB1 by inserting a light sensitive protein module in the EB1 protein. This module consists of the plant light receptor domain LOV2, plus a a small protein that binds LOV2 in the dark but dissociates in the presence of blue light. Below you find a schematic of photo-inactivated(pi)-EB1, and images of a cell before and after inactivation of pi-EB1. Click here to go to the publication


pi-EB1

Schematic of pi-EB1(left) and example of pi-EB1 inactivation and reversion in a human cell

We also published supporting protocols on how to make cell lines expressing this optogenetic switch, and provide some advice on things to keep in mind when designing your own optoswitches using this "domain splitting" strategy.
Click here for the supporting protocols

pi-EB1 constructs are available on Addgene
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We recently published a chapter in Methods in Molecular Biology, where describe some of the ins and outs of cellular optogenetics experiments, with a focus on combining optogenetics and high resolution microscopy.
This chapter focusses mostly on the use of LOV2 based optogenetics switches. We also discuss how to locally control optoswitches in cells through the use of a Digital Micromirror Device or DMD(Mightex Polygon), and we describe a DIY blue LED cube that can be used to illuminate 2 wells of a 12 well glass bottom plate (and which fits inside small environmental chambers used on many microscopes), and is controlled by an Arduino.
Click here to go to the Methods chapter


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In the near future i'll try to upload more detailed step-by-step instructions on how to assemble the LEDcube

For more info also see:
- Our work was highlighted on: the Addgene blog.
- Mightex application note:"Spatial control of microtubule dynamics at the subcellular scale by patterned illumination"

This website is still very much a work in progress!
https://optogen.net, all material published on this website is copyrighted. For info contact: Jeffrey at optogen dot net