Fig. 3: Design and characterisation of circuit TKS2.

a Schematic representation of the circuit TKS2. Blunt arrows indicate repression and pointy arrow activation. b Schematic representation of relevant DNA segments of the MTncat with TKS2 used to generate a polyclonal strain (chloramphenicol used for selection). White triangles for the inverted repeats (IR); Bend-arrows for constitutive promoters (black) and ATc-inducible (white) promoters. Arrows for genes (grey); Black semicircles show the RBSs; A white square for the riboswitch (RS); Black triangles indicate the position and orientation of the lox sites (lox66 and lox71); cat, selection cassette. Blue brackets indicate the segment that is duplicated in the analysis of the circuit with 2xTetR cassette. c DNA gel showing the results from the PCR analysis of Cre/lox recombination in the TKS2 from genomic DNA samples from passages 1 and 2 in theophylline medium with Cre basal expression (p1+ and p2+) and passage 2 in theophylline-free medium and Cre induction (p2-). PCR product size is expected for the full fragment, before Cre/lox recombination, and the distance between lox sites are indicated in (b) between the small head arrows S5 and S17, showing the position of the primers pair used in the assay (in blue for 2xTetR). d Western blot results showing TetR, CI and Cas9 expression in strains with the circuit TKS2. Expression of the proteins was analysed using 10 µg of total protein extracts from incubations of the strains in the growth permissive condition of 0.5 mM theophylline (+); after a single passage in theophylline-free medium producing slow activation of the TKS2 (−); after two passages in theophylline-free medium (2−) or in 50 ng/mL of ATc to trigger the kill-switch (ATc). Results presented from the circuit engineered with one or two copies of the pSpi-riboswitch-tetR cassette between the lox pair sites in the circuit (1xTetR or 2xTerR, respectively). DNA gel and western blot results are representative of two independent experiments in each case.