3.3 Efficient creation of marker-free transplastomic lines using
CpPosNeg
To validate the CpPosNeg system (Figure 1 ), plasmids were
designed in which the dual marker was placed downstream of lucCP ,
a codon-optimized reporter encoding firefly
luciferase.[42] For both gene cassettes, the same
3’ UTR element from rbcL was used in order to create a 258 bp
direct repeat (see: Supplementary Figure 1) . This size
of repeat was chosen since it is significantly smaller than the 462 bp
needed for high rates of intramolecular recombination in the absence of
selection in the C. reinhardtiichloroplast,[24] but larger than the minimum size
(~210 bp) reported for such recombination to
occur.[26] The dual marker was therefore predicted
to be stably maintained in the intermediate transformant lines (R1) in
the absence of Spc selection, but efficiently lost in the R2 lines
following counter-selection on 5-FC (Figure 1b ). Left and right
homology arms of ~1000 bp were placed upstream and
downstream of the reporter and marker cassettes in order to target the
genes to two different neutral insertion sites: either downstream ofpsbA [39] (plasmid pLuc1) or downstream ofpsbH [43] (plasmid pLuc2).
WT C. reinhardtii was transformed using pLuc1 or pLuc2 with
selection based on Spc resistance conferred by the dual marker. For each
transformation, six colonies were re-streaked on Spc over three
generations to drive the cells to the R1 homoplasmic state
(Figure 1d ). As illustrated in Figure 4a , a
four-primer multiplex PCR analysis of either the psbA locus or
the psbH locus was employed to confirm the genotype with
diagnostic band sizes for the WT, R1 and R2 loci. All six transformant
lines showed the R1 genotype and appeared to be homoplasmic following
three rounds of Spc selection, with no detection of the WT band.
Importantly, two further rounds of replating on medium lacking Spc
demonstrated that the codA-aadA marker DNA was stably maintained
in the plastome despite being flanked by the rbcL direct repeat,
with the PCR analysis showing maintenance of the R1 band and no
appearance of the R2 band (Figure 4c ). Conversely, two rounds
of plating on 5-FC medium led to the rapid loss of the marker, with the
R2 plastome appearing to be homoplasmic since only the PCR product from
primers P2 and P4 was detected (Figure 4d ). Sequencing of this
PCR product confirmed the loop-out of the marker at both the psbAand psbH loci via recombination between the rbcL copies.
An assay of luciferase activity in the R1 and R2 lines confirmed that
the introduced lucCP cassette was expressed and that the level of
expression was not affected by the subsequent loop-out of the cassette
with both pairs of R1 and R2 lines showing similar activities
(Figure 5e ). There was a small but significant difference in
expression in Luc1.R2 relative to Luc1.R1 in the conditions tested
(3.6%; p = 0.03; Student’s t -test). While this could be
due to the removal of the selection cassette, it is more likely an
artefact due to subtle differences in the culture history and incubation
conditions of the samples. There was no significant difference between
expression in Luc2.R1 and Luc2.R2 (p = 0.13; Student’st -test). Interestingly, the targeting of lucCP into the
plastome’s large inverted repeat region downstream of psbA(transformant Luc1) such that two copies are present per plastome
molecule rather than one as in the case of the psbH transformants
(Luc2) gave more than twice the luciferase activity. This suggests that
the activities of the rrnS promoter and psaA 5’UTR
elements used to drive lucCP expression are not limiting, and
that the level of recombinant protein is directly related to copy
number. This is a surprising finding given that copy number is assumed
not to be a key factor in chloroplast
expression[44] and that transgene expression is
mainly controlled at the translational level.[45]However, we cannot rule out genomic context and the influence of
upstream/downstream transcriptional units as an alternative explanation
for the different expression levels.
After removal of the codA-aadA cassette, the R2 phenotype should
be the same as the WT with respect to sensitivity to Spc and resistance
to 5-FC. To confirm this, spot tests were carried out on WT, Luc1.R2 and
Luc2.R2 (Figure 5f ). C-A.f (Spc resistant; 5-FC sensitive) was
included as a positive control. Luc1.R2 and Luc2.R2 showed the same
phenotype as WT: full dieback on Spc and similar levels of growth in the
presence or absence of 5-FC. This further confirmed that thecodA-aadA marker had been completely lost in the R2 cell lines.