Fig. 3: Benefits of IBM and reduction of basal activities by IBM.

a Substitution of the split M86 intein inserted in split TetR by SYNZIP. Representative sites were chosen within the three identified split seams. Results showed different split sites had differential tolerances towards additional protein domains, but all split sites functioned well when the intein was used (Fig. 2b). Single-cell fluorescence values were pooled from three biological replicates (n = 3). b IBM-identified split sites of homologous TetR and SrpR do not necessarily map to loops between consensus helical domains, highlighting the limitation of guessing split sites for a new protein by secondary structure alignment. HTH helix-turn-helix. c Splitting highly active proteins can reduce their basal activities. Left panel: leaky expressions of β-lactamase (BLA) led to ampicillin resistance in the absence of induction, which could be improved by splitting BLA. Middle and right panels: fluorescence distributions and fold changes between fully on and fully off states of intact and split TetR and ECF20 were shown. Representative sites were chosen from each identified seam. In most cases the split clones had lower basal activities and therefore larger fold changes between on and off states. Single-cell fluorescence values were pooled from experiments performed on three different days (n ≥ 3, see Supplementary Data 2, sheet “sample_sizes” for exact values of n). DAPG 2,4-Diacetylphloroglucinol. b, c Data reused from Fig. 2 and Supplementary Fig. 8 but further analyzed. a, c In fold change calculations, bar heights and error bars represent mean and SD.