FIG. 1.

Schematic representations of NAD biosynthesis pathways and principles for the establishment of an antibiotic-free plasmid selection system based on complementation of host auxotrophy in the NAD de novo pathway. (A) NAD synthesis pathways in vertebrates and E. coli. The red arrows show the steps in vertebrates, the blue arrows show the steps in E. coli, and the black arrows show the common reactions. (B) A ΔQAPRTase E. coli strain was constructed, and E. coli was able to survive, even when the NAD de novo pathway was blocked, in a medium containing an NAD precursor. (C) ΔQAPRTase E. coli could not survive in media, such as M9 medium, that lacked an NAD precursor. (D) The ΔQAPRTase E. coli strain, which was auxotrophic in the NAD de novo pathway, could be complemented with the plasmid-borne selection gene.