Light-dependent Control of Bacterial Expression at the mRNA Level

Abstract

Sensory photoreceptors mediate numerous light-dependent adaptations across organisms. In optogenetics, photoreceptors achieve the reversible, non-invasive, and spatiotemporally precise control by light of gene expression and other cellular processes. The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination. By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest, their expression can be downregulated by light. We developed the pCrepusculo and pAurora optogenetic systems that are based on PAL and allow to down- and upregulate, respectively, bacterial gene expression using blue light. Both systems are realized as compact, single plasmids that exhibit stringent blue-light responses with low basal activity and up to several ten-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that generally control transcription initiation. By integrating regulatory mechanisms operating at the DNA and mRNA levels, optogenetic circuits with emergent properties can thus be devised. As a case in point, the pEnumbra setup permits to upregulate gene expression under moderate blue light whereas strong blue light shuts off expression again. Beyond providing novel signal-responsive expression systems for diverse applications in biotechnology and synthetic biology, our work also illustrates how the light-dependent PAL-aptamer interaction can be harnessed for the control and interrogation of RNA-based processes.

Optogenetic Control of Bacterial Expression by Red Light

“…Against this backdrop, we cloned the YPet fluorescent protein into pREDusk and pREDawn and combined the resultant plasmids with the pCrespusculo- Ds Red and pAurora- Ds Red systems that are based on the RNA-binding LOV receptor PAL 11 and respond to blue light. 66 Briefly, pCrepusculo encodes PAL, which upon activation by blue light specifically binds to a short RNA aptamer. Said aptamer is embedded near the ribosome-binding site of an mRNA encoding Ds Red.…”

“…Although multimodal optogenetic control was established in bacteria before, , it often required the elaborate optimization of plasmids and strains. Against this backdrop, we cloned the YPet fluorescent protein into pREDusk and pREDawn and combined the resultant plasmids with the pCrespusculo- Ds Red and pAurora- Ds Red systems that are based on the RNA-binding LOV receptor PAL and respond to blue light . Briefly, pCrepusculo encodes PAL, which upon activation by blue light specifically binds to a short RNA aptamer.…”

“…First, as most setups for light-regulated bacterial expression respond to shorter wavelengths, predominantly in the blue spectral region, pREDusk and pREDawn lend themselves to be orthogonally addressed and jointly used with these systems. We evaluated this concept by combining pREDusk and pREDawn with the pCrepusculo and pAurora plasmids 66 that encode the LOV receptor PAL and downregulate and upregulate, respectively, the expression in response to blue light ( Figure 5 ). As expected, based on the action spectra of LOV receptors, 11 neither pCrepusculo nor pAurora was toggled by red light.…”

“…The combination of the red-light-responsive pREDusk-YPet and pREDawn-YPet with the blue-light-responsive pCrepusculo and pAurora setups 66 was studied likewise with the following modifications to the protocol. To address the systems separately, the pCrepusculo and pAurora systems were equipped with the Ds Red fluorescent reporter.…”