FluOpti: an open-source system for programmable whole-plate optogenetics and multi-fluorescence imaging

Abstract

Bacteria are able to self-organize into complex spatial communities, such as colonies, biofilms and swarms. Probing cells and gene networks with dynamic stimuli while preserving their spatial organization is therefore essential for understanding how bacterial communities organize across spatial and temporal scales. Optogenetics offers a powerful approach for such a task given the precise control of stimulus delivery without the perturbations normally associated with the addition of liquid inducers. However, most optogenetics devices have been developed to work with liquid cell cultures or to deliver a spatial pattern without the concurrent recording of gene responses and colony growth. Here, we describe FluOpti, an open source, low-cost device for controlling gene expression, through programmable light inputs, and recording multi-fluorescence responses and colony growth across whole petri dishes. White light LEDs are used for brightfield imaging while green and red LEDs are used as optogenetic inputs to activate and inactivate target genes via the CcaS/R two component system in E. coli. Low cost blue and orange acrylic are used as excitation and emission filters for fluorescence imaging, respectively. We developed custom software to analyze colony growth and optogenetic responses from 72-hour time-lapse experiments. A mathematical model was developed to predict gene expression levels in response to green-to-red light ratio stimuli. We use FluOpti to reveal a spatially structured response to optogenetic stimuli, with gene activation rates differing in cells located at the center and edge of colonies. to validate its use at a whole petri dish scale, FluOpti was used to depict how Voronoi territories can act as predictors of colony size, in congruence with previous studies. Together, these results demonstrate the utility of the device for studying gene networks at multiple spatio-temporal scales, from individual colonies to the whole petri dish. Performing optogenetics studies in whole petri dishes while preserving spatial arrangements could offer valuable insights into the spatial biology of gene networks in microbial populations.