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Plant Cell Physiol. 2013 Oct;54(10):1724-35. doi: 10.1093/pcp/pct115. Epub 2013 Aug 21.

Theophylline-dependent riboswitch as a novel genetic tool for strict regulation of protein expression in Cyanobacterium Synechococcus elongatus PCC 7942.

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Venture Business Laboratory, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime, 790-8577 Japan.


The cyanobacterium Synechococcus elongatus PCC 7942 is a major model species for studies of photosynthesis. It is are also a potential cell factory for the production of renewable biofuels and valuable chemicals. We employed engineered riboswitches to control translational initiation of target genes in this cyanobacterium. A firefly luciferase reporter assay revealed that three theophylline riboswitches performed as expected in the cyanobacterium. Riboswitch-E* exhibited very low leaky expression of luciferase and superior and dose-dependent on/off regulation of protein expression by theophylline. The maximum magnitude of the induction vs. basal level was ∼190-fold. Furthermore, the induction level was responsive to a wide range of theophylline concentrations in the medium, from 0 to 2 mM, facilitating the fine-tuning of luciferase expression. We adapted this riboswitch to another gene regulation system, in which expression of the circadian clock kaiC gene product is controlled by the theophylline concentration in the culture medium. The results demonstrated that the adequately adjusted expression level of KaiC restored complete circadian rhythm in the kaiC-deficient arrhythmic mutant. This theophylline-dependent riboswitch system has potential for various applications as a useful genetic tool in cyanobacteria.


Circadian clock; Cyanobacteria; Regulation of gene expression; Riboswitch; Synechococcus elongatus PCC 7942; Theophylline

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