Characterizing Optogenetic Tools for Use in Synthetic Gene Circuits.

Synthetic gene circuits consist of engineered networks of genes and proteins. The function of these circuits can be tuned by regulating the expression levels of key circuit components. Light is an ideal signal for controlling expression levels in these circuits; it is cheap and easy to modulate relative to traditional chemical signals. Here, we explore Optogenetic Transcription Factor Localizers (OpTFLos) for tuning expression levels with light. OpTFLos are genetically encoded light-responsive tools for controlling the position in the cell, and therefore the activity, of transcription factors. In this protocol, we discuss how to use the automated optogenetics platform Lustro to systematically characterize OpTFLos to identify light programs that generate desired expression levels. One challenge here with optogenetic tools is that many systems respond to blue light. By using microscopy and Lustro, we describe how to identify light programs that allow for two blue-light-responsive OpTFLos with different kinetic properties to achieve distinct gene expression states with minimal crosstalk, potentially useful for tuning the expression of multiple components within a synthetic gene circuit with a single wavelength of light.