Figure 1: Marker recycling strategy. (A, C )
Transformants are generated by integration of the CpPosNeg plasmid into
a neutral site within the WT plastome via homologous recombination of
left and right homology arms (LHA and RHA) and selection on Spc
conferred by aadA. Initial transformants are then re-streaked on plates
supplemented with Spc to eliminate all WT copies and generate the
intermediate R1 plastome. (B, D ) The R1 strains are then
re-streaked on plates supplemented with 5-FC. Negative selection
conferred by codA selects for plastome copies where thecodA-aadA marker has been lost via intramolecular recombination
of the rbcL direct repeat. Re-streaking on 5-FC results in the
final marker-free R2 strain.