Engineering gene overlaps to sustain genetic constructs in vivo
Fig 4
Synthesized construct and experimental design.
(a) Synthetic construct: overlapping reading frame between galK, which confers sensitivity to DOG, and kanR, which confers resistance to kanamycin. The expression of both genes is controlled by the pLac promoter: in absence of the IPTG inducer, the operon is repressed by lacIq. (b) galK metabolises DOG into a toxic compound (see S4 Fig for more details). (c) Experimental design: random mutations appear during growth in absence of selection and are subsequently scored by plating on selective medium. This design permits to test whether purifying selection applied on kanR (presence of kanamcyin) protects galK from loss-of-function mutations.