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 endogenoustscA –chlN 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.