3.1 The CpPosNeg marker strategy
The CpPosNeg marker-recycling strategy is divided into two recombination events, which we call R1 and R2 (Figure 1 ), leading to the final unmarked strain. Initially, integration of the CpPosNeg plasmid into the C. reinhardtii plastome occurs via intermolecular recombination between the ~1000 bp left and right homology arms (LHA and RHA) of the plasmid and the corresponding regions of the plastome. Selection on spectinomycin (Spc) allows generation of an insertional cell line containing both the GOI cassette and the positive/negative selection cassette encoding the CodA-AadA fusion protein. Cell lines are serially re-streaked on Spc to eliminate any WT copies of the plastome. Depending on growth regimes, the chloroplast has on average ~ 83 copies of the plastome,[13] and all copies within the chloroplast must contain the R1 DNA in order to proceed with the strategy and avoid reversion to the WT genotype following removal of the Spc selection pressure. In the second recombination step, cells are treated with 5-FC thereby imposing a negative selection pressure for retention of the CodA activity. This selects for loss of the dual marker cassette from the plastome via intramolecular recombination between the two copies of the rbcL 3’ UTR element linked to the marker and the GOI, respectively.