Abstract
Optogenetic systems utilize genetically encoded light-sensitive proteins to control cellular processes such as gene expression and protein localization. Like most synthetic systems, generation of an optogenetic system with desirable properties requires multiple design-test-build cycles. A yeast optogenetic toolkit (yOTK) allows rapid assembly of optogenetic constructs using Modular Cloning, or MoClo. In this protocol, we describe how to assemble, integrate, and test optogenetic systems in the budding yeast Saccharomyces cerevisiae. Generating an optogenetic system requires the user to first define the structure of the final construct and identify all basic parts and vectors required for the construction strategy, including light-sensitive proteins that need to be domesticated into the toolkit. The assembly is then defined following a set of standard rules. Multigene constructs are assembled using a series of one-pot assembly steps with the identified parts and vectors and transformed into yeast. Screening of the transformants allows optogenetic systems with optimal properties to be selected.