Fig. 3
Engineering of the genome-integrated farnesyl diphosphate synthase (FPPS) and disruption of farnesyl diphosphate phosphatase (YisP) in B. subtilis. (a) pJOE8999.g_sigX_fpps was designed to allow the replacement of the sigX gene from B. subtilis by the fpps gene from B. megaterium under the control of a strong promoter PsigX. (b) pJOE8999.g_ΔyisP was constructed for the disruption of the yisP gene from B. subtilis (c) Upon transformation, the resulting B. subtilis strain harboring both genomic modifications along with three gene-copies of the crtMN genes under the control of the constitutive promoter PsigX was designated as BsMN6. (d) Confirmation of the sigX gene replacement by fpps in the BsMN5 strain. Lane 1 corresponds to an amplification band of 2.5 kb using primers P2F/P4R to verify fpps integration at the sigX locus site in BsMN5. Lane 2 corresponds to an amplification band of 2.25 kb using the same primers in recipient strain BsMN3. M corresponds to the molecular marker weight. (e) Confirmation of the yisP gene disruption in strain BsMN6. Lane 1 corresponds to an amplification band of 1.75 kb using primers P5F/P6R to verify yisP deletion in BsMN6. Lane 2 corresponds to an amplification band of 2.5 kb using the same primers in recipient strain BsMN5. M corresponds to the molecular marker weight