Extended Data Fig. 4: Robust, tunable, and modular SENTR for gene regulation.

a, Trans-splicing performance for 16 base pair combinations at 5′ splice site. b, Choices of 5′ splice sites inside 5′ UTR (site −6 and −1) or start codon (site 2). c, Design schematic of mRNA sensor. The mRNA binds to 3′ EGS to repress the 3′ RNA, thus lowering the trans-splicing fluorescence. d, Transfer function for the mCherry sensor (blue curve, left y-axis) and mCherry (purple curve, right y-axis) as a function of rhamnose concentration. The sensor expression was induced by 3.2 µM AHL, and mCherry expression was induced by rhamnose. e, Splice site selection and performance for sgRNA trans-splicing. Combinations of RNA strands were achieved via the induction (+) and non induction (-) of certain RNA expression, and absence (∅) of certain RNA generator in circuits. The active (+) and deactivated (-) introns were used for assaying the sgRNA activity without trans-splicing reactions. Inset, sgRNA design and 5′ splice site. f, Genetic design architecture of modular SENTR. Yellow rectangles, split intron halves. Orange rectangles, EGSs. g, Tuning modular SENTRs by seven RBSs and four induction levels of 5′ RNAs. h, Modular SENTR from SunY intron. The split SunY intron, with its native junction sequences, was inserted into codon 1 of sfGFP CDS. Data in (a, d-e, g-h) were collected by flow cytometry. For (a, g), the heat maps show mean values of n = 3 biological replicates. For (e, h), bars show mean values and error bars represent s.d. of n = 3 biological replicates. For (d), the points show mean values and error bars represent s.d. of n = 3 biological replicates. Bacteria transformed with empty vectors and J23101-sfGFP/mCherry were used as negative and positive controls for calculating fluorescence values in relative promoter units (RPU).