Curated Optogenetic Publication Database

Abstract: Multiple display techniques, including phage display, mRNA display, and ribosome display, have been used to expand the optogenetic toolbox beyond what nature provides. These techniques are most often applied to the development of binding partners that selectively recognize different conformational states of photoswitchable proteins. However, for some targets, in particular the spectrally diverse cyanobacteriochrome (CBCR) GAF domain family, the subtle differences between conformational states pose a significant challenge to discovering highly selective binders. We present an optimized phage display-based protocol designed to effectively capture these subtle changes. This optimized protocol applies high selection pressure by changing the elution method and tightening negative selection, leading to the enrichment of selective binders. Through multiple selection campaigns, we demonstrate the utility of this protocol for identifying highly selective binders.

Abstract: Optogenetics has emerged as a powerful tool for regulating cellular processes due to its noninvasive nature and precise spatiotemporal control. Far-red light (FRL) has increasingly been used in the optogenetic control of mammalian cells due to its low toxicity and high tissue penetration. However, robust and orthogonal FRL sensors are lacking in bacteria. Here, we established an orthogonal FRL sensor in Escherichia coli with a maximum dynamic range exceeding 230-fold based on the RfpA-RfpC-RfpB (RfpABC) signaling system that regulates the far-red light photoacclimation (FaRLiP) in cyanobacteria. We identified a conserved DNA motif in the promoter sequences of the Chl f synthase gene and other genes in the FaRLiP gene clusters, termed the far-red light-regulatory (FLR) motif, which enables the light-responsive activation of gene expression through its interaction with RfpB. Based on the FLR motif, we simplified the FLR-containing promoters and characterized their activation abilities and dynamic ranges, which can be utilized in different synthetic biology scenarios. Additionally, one or two FLR motifs are present at other loci within the FaRLiP gene cluster, providing further FRL-inducible promoter resources. The FRL sensor exhibits effective activation and suppression under low-intensity FRL and white light, respectively, and remains functional in darkness. In conclusion, this study advances the understanding of the regulatory mechanisms of FaRLiP in cyanobacteria and provides robust and orthogonal FRL sensors for synthetic biology applications.