Programming the Dynamic Control of Bacterial Gene Expression with a Chimeric Ligand- and Light-Based Promoter System

Abstract

To program cells in a dynamic manner, synthetic biologists require precise control over the threshold levels and timing of gene expression. However, in practice, modulating gene expression is widely carried out using prototypical ligand-inducible promoters, which have limited tunability and spatiotemporal resolution. Here, we built two dual-input hybrid promoters, each retaining the function of the ligand-inducible promoter while being enhanced with a blue-light-switchable tuning knob. Using the new promoters, we show that both ligand and light inputs can be synchronously modulated to achieve desired amplitude or independently regulated to generate desired frequency at a specific amplitude. We exploit the versatile programmability and orthogonality of the two promoters to build the first reprogrammable logic gene circuit capable of reconfiguring into logic OR and N-IMPLY logic on the fly in both space and time without the need to modify the circuit. Overall, we demonstrate concentration- and time-based combinatorial regulation in live bacterial cells with potential applications in biotechnology and synthetic biology.

Engineering an Optogenetic CRISPRi Platform for Improved Chemical Production

“…Because of its spatiotemporal control, optogenetic regulation has recently been developed and applied in the field of synthetic biology. ,, In this study, we sought to construct a blue light-responsive repression system based on EL222 as previously described. , EL222 is a light-sensitive protein which has a light-oxygen-voltage (LOV) domain that senses blue light signals and a DNA-binding domain (DBD) that binds DNA sequences. EL222 binds DNA in the form of homodimer, the excitation time and reversion time of which are in seconds. , EL222 has been developed as an optogenetic switch for gene expression regulation, and its derivatives have also been optimized as transcriptional activators and repressors. ,, …”

“…Because of its spatiotemporal control, optogenetic regulation has recently been developed and applied in the field of synthetic biology. 25,30,31 In this study, we sought to construct a blue light-responsive repression system based on EL222 as previously described. 32,33 EL222 is a light-sensitive protein which has a light-oxygen-voltage (LOV) domain that senses blue light signals and a DNA-binding domain (DBD) that binds DNA sequences.…”

A Timed Off-Switch for Dynamic Control of Gene Expression in Corynebacterium Glutamicum

“…While these systems are usually easy to handle and well-suited for lab scale studies, their application in the industrial environment is often hampered by, e.g., the price or availability of the effector molecule (Ferreira et al, 2018 ; Cardoso et al, 2020 ). Moreover, since most effector molecules show a high stability and cannot easily be removed from the culture broth, dynamic control of gene expression is still challenging and therefore subject of recent studies in different organisms (Jayaraman et al, 2018 ; Baumschlager et al, 2020 ; Wiechert et al, 2020 ; Glasscock et al, 2021 ).…”

BMSS2: a unified database-driven modelling tool for systematic model selection and identifiability analysis

“…Modelling in synthetic biology guides experimental designs and enables rational optimization of the performance of biological systems (Elowitz, 2000; Jayaraman et al, 2016). In particular, kinetic modelling offers quantitative insights into the dynamic aspects and interactions of the systems components (Jayaraman et al, 2018). This has empowered researchers to improve the designs of genetic devices including biosensors (Holowko et al, 2016), cell factories (Yeoh et al, 2020), therapeutics (Caliendo et al, 2019), and integral feedback systems (Aoki et al, 2019).…”