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.