Fig. 2: Design and characterisation of the kill-switch KS1.
From: A genetic toolkit and gene switches to limit Mycoplasma growth for biosafety applications
a Schematic representation of the kill-switch KS1. Blunt arrows indicate repression and pointy arrow activation. b Schematic representation of relevant DNA segments of the MTnpar with the KS1 used to generate a polyclonal strain (puromycin used for selection). White triangles represent the inverted repeats flanking the fragment of the MTn inserted in the genome of Mycoplasma (IR); Bend-arrows for constitutive promoters (black) and IPTG-inducible (white) promoters. Arrows for genes (grey), gRNA (stripped; gRNA2 has eight targets in the genome of M. pneumoniae); par, selection cassette. c Western blot results showing Cas9 expression in strains with the KS1 kill-switch with the cas9 gene with the canonical initiation codon ATG (I) or a CTG codon instead (II). Protein expression was analysed using 10 μg of total protein extract from pre-induced (−) and 1 mM IPTG-induced (IPTG) samples. d Growth kinetics of polyclonal strains with KS1 without any gRNA (I), with a single gRNA copy (II) and same but pre-treated with IPTG in the previous passage (III). Graphs show growth kinetics (growth index corresponding to the ratio Abs430nm/Abs560nm) when KS1 is uninduced (0 mM IPTG, blue lines) or IPTG-induced (kill-switch activated with 1 mM IPTG, red lines). Mean values from three bio-replicates. Error bars indicate standard deviation. Source data are provided as a Source Data file.
