Blue light-mediated transcriptional activation and repression of gene expression in bacteria

Abstract

Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes.

An inducible AraC that responds to blue light instead of arabinose

“…Moreover, since we constructed pBLADE using pBAD33 as template ( Supplementary Fig. 2a While many other light-inducible TFs have been developed to date 61,[68][69][70][71][72] , some of which featuring extremely high dark/light fold changes 61, 72 , we explicitly aimed to engineer a system based on a well-known and pervasive TF (namely AraC) that is particularly suited for microbiological applications, thus stimulating the use of optogenetics in microbiology. We took special care to engineer BLADE with minimal leakiness.…”

Controlling Cells with Light and LOV

“…An interesting, recent example for a naturally occurring, light‐dependent transcriptional regulator is EL222 from E. litoralis , which binds a cognate DNA motif upon light‐mediated homodimerization. By transplanting its DNA binding motif to engineered promoters, EL222 can be used to control gene expression in E. coli and mammalian cells (using an EL222–VP16 fusion) with high spatiotemporal precision . Zhao et al have recently also applied EL222–VP16 to improve the production of different, energy‐rich alcohols in engineered yeast .…”

Illuminating pathogen–host intimacy through optogenetics

“…As depicted in Fig 1A and [13,14], cGMP [15,16], or c-di-GMP [42,43]. It is also possible to develop light-responsive expression systems using photocaged effectors, such as doxycycline [44] and IPTG (isopropyl β-D-1-thiogalactopyranoside) [45] or proteins [46,47], which are released or activated upon illumination to control the gene expression or protein activity. Just recently, the LOV2-ODC-degron system has been reported, which targets the conjugated protein of interest to light-dependent proteasomal degradation, thereby controlling the protein stability [48] (Fig 1B).…”