A Logic NAND Gate for Controlling Gene Expression in a Circadian Rhythm in Cyanobacteria

Abstract

To enable circadian control of gene expression in cyanobacteria, we constructed a genetic logic gate (NAND) using orthogonal promoters via modular CRISPR interference. The NAND gates were tested in Synechococcus elongatus PCC 7942 using a fluorescent reporter. The NAND gate dynamics were characterized based on the affinity of the dCas9 complex to the output element. Upon connecting tight gene repressions with the circadian promoter (the purF gene; peak expression at dawn), inversed peak expressions were obtained as an output of the NAND gate although the retroactivities were shown in the ON and OFF states. A dark-responsive genetic element of the NAND gate was also expanded to an AND gate in S. elongatus PCC 7942. These cyanobacterial NAND and AND gates could facilitate the control of gene expressions in dynamic metabolic engineering technologies, thereby enabling the cyanobacteria to serve as biosolar cell factories.

Sustainable citric acid production from CO2 in an engineered cyanobacterium

“…Further iterations of the current strains could test constitutive promoter libraries ( Markley et al, 2015 ) to express aconitase at the right level to balance these two constraints. Alternatively, a more dynamic control system—for instance, by integrating both 2-oxoglutarate and citrate levels through a NAND gate ( Lee and Woo, 2020 ) to control aconitase output—could prove more useful than a constitutive promoter, by continuously adapting flux restriction to cellular metabolic requirements. Additionally, future strain iterations should also aim to improve flux toward oxaloacetate and/or acetyl-CoA as this may also increase production titers, by improving the available pools of both precursors.…”

Current Metabolic Engineering Strategies for Photosynthetic Bioproduction in Cyanobacteria

“…The use of CRISPRi as a modular and flexible gene regulation system that responds to different stimuli was recently showcased by combining it with two physically and chemically inducible promoters [191]. The synthetic systems used the circadian clock as a blueprint for a genetic circuit that computes an NGATE logic.…”

“…The designed genetic circuit was composed of YFP controlled by the IPTG-inducible promoter Ptrc, a sgRNA targeting Ptrc, driven by a dark-induced promoter Purf and a dCas9 controlled by the aTc-inducible promoter Ptet. In the presence of IPTG, downregulation of YFP is only achieved in the presence of the two inputs: darkness and aTc addition; light conditions, instead, lead to deactivation of Purf and derepression of YFP [191].…”

Engineering a Controllable Targeted Protein Degradation System and a Derived OR-GATE-Type Inducible Gene Expression System in Synechococcus elongatus PCC 7942

“…In recent years, genetic tools that allow controllable gene expression, including sensor elements and inducible promoters, − ribo-switches, , small RNA (sRNA) regulatory tools, and CRISPRi systems, − have been developed and used in cyanobacteria metabolic engineering, facilitating dynamic regulation of the target proteins synthesis rates on the stages of transcription and translation. On the basis of these approaches, complex genetic circuits responsive to diverse artificial, environmental, or physiological signals have also been developed. ,− However, in contrast to the toolboxes developed for more typical microbial chassis such as Escherichia coli and Bacillus subtilis , what is relatively missing from dynamic regulation in cyanobacteria is the controllable protein degradation system that allows for targeted degradation of specific proteins and manipulation of the protein concentrations. − With respect to the approaches that regulate the protein synthesis rates, the target protein that has been synthesized would remain in the cytoplasm before being naturally digested or diluted during cell growth and replications. , In contrast, protein degradation approaches could allow for a rapid decrease in protein concentrations independent of cell proliferation events, enabling a rapid metabolic response to environmental or artificial signals. Thus, controllable protein degradation systems would be an attractive option for expanding the cyanobacteria genetic toolbox.…”