3.2 Development of a codA-aadA dual marker through translational fusion
Whilst both markers have individually been shown to be functional in theC. reinhardtii chloroplast,[19,32] and AadA has been shown to retain functionality when synthesised as a C-terminal fusion to endogenous chloroplast proteins,[37] the creation of a dual marker conferring both Spc resistance and 5-FC sensitivity has not been demonstrated previously. We therefore created two initial plasmid constructs in which codA and aadA were fused together, either at the transcriptional or the translational level (Figure 2 ). In plasmid pC-A the coding sequences are linked via a flexible linker sequence (encoding GGSGGGSG[38]) to create a single CodA-AadA fusion protein. In pC-IEE-A the two coding sequences are transcriptionally linked as an biscistronic operon via an intercistronic expression element (IEE) derived from the endogenoustscAchlN intergenic region.[39]For these initial constructs, direct repeat elements were not included so that the marker genes would remained stably integrated in the plastome.
Homoplasmic transformant lines were recovered for both constructs following biolistic transformation of the WT strain (Figure 2b ). Phenotypic tests were then carried out by spotting cultures on selective medium. For both classes of transformant, the dual functionality of the marker was confirmed by their ability to grow on Spc and inability to grow on 5-FC, in contrast to the untransformed WT strain (Figure 2c ).
Since both arrangements of the codA and aadA coding sequence gave very similar phenotypes, it was decided to take the translational fusion forward since this avoided introducing a duplicate copy of the IEE into the plastome, which might promote unwanted recombination between the marker and the tscA-chlN locus. However, since fusing CodA and AadA might compromise the efficient folding of either enzyme moiety, and hence full enzyme activity, we tested two further linkers with respect to transformation efficiency and acquired sensitivity to 5-FC. In addition to the original flexible linker GGSGGGSG, the two proteins were connected via either a rigid helix-forming linker (LAEAAAKEAAAKAAA[40]) designed to give spatial separation of the two enzymes, or the short linker ISGANGV.[38] All three constructs yielded Spc-resistant colonies following chloroplast transformation of the WT strain, but transformation efficiencies with the rigid and short linker constructs were seen to be much lower than those obtained with the flexible linker (Figure 3b ). We concluded that the flexible linker was the most optimal for AadA activity, and it is likely that this is beneficial for Spc selection in the initial phase of transformation when only a few plastome copies carry the marker.[41] CodA activity also appeared to be higher when fused via the flexible linker since spot tests showed greater sensitivity to 5-FC when compared to transformants generated using the rigid or short linker constructs (Figure 3d ). In light of these results, the dual marker with flexible linker (C-A.f) was selected for construction of the CpPosNeg plasmids.