BioDesign Research / 2020 / Article / Fig 3

Research Article

Engineering a Circular Riboregulator in Escherichia coli

Figure 3

Characterization of activation of gene expression by the engineered circular riboregulator. (a) Scheme of the construction used: the riboregulator circRAJ31 can trans-activate a cis-repressed gene coding for sfGFP with a degradation tag (LAA). IPTG induces production of the mRNA and aTc of the ribozyme RNA, which releases the circular riboregulator after self-splicing. (b) Scheme of the control systems constructed: the toehold mutant (to inactivate interaction), the C873U mutant (in intron 2, to inactivate self-splicing), and the linear riboregulator RAJ31min (without the T4-PIE ribozyme scaffold). (c) Characterization of systems based on circRAJ31, the native system, and its controls. See also the effect of different concentrations of IPTG and aTc in Figure S5. (d) Scheme of the construction used: the riboregulator circRAJ31 can trans-activate a cis-repressed cat gene, which codes for chloramphenicol resistance (CamR). (e) Characterization of circRAJ31 controlling growth of E. coli on chloramphenicol plates (35 μg/mL), with appropriate inducers of gene expression (see the plates in Figure S7). (f) Characterization of circRAJ31 and the C873U mutant controlling growth of E. coli on liquid cultures (70 μg/mL chloramphenicol), with appropriate inducers of gene expression (this plot is extended in Figure S8). In all cases, error bars indicate standard deviations.
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