Figure 2: Validation of codA and aadAfunctionality as translational or transcriptional fusions. (A )
Design of the translational and transcriptional plasmids pC-A and
pC-IEE-A respectively. LHA/RHA; left and right homology arms for
targeting to a neutral locus downstream of psbA. The promoter and 5’UTR
elements used to drive codA/aadA expression are from atpA, and the 3’UTR
from rbcL . IEE; tscA-chlN intercistronic expression
element. (B ) Three primer PCR strategy for checking homoplasmic
transformants. The wild-type (WT) plastome yields a 2455 bp product with
primers P1 and P2, whereas the recombinant (Rec.) plastome gives a 2078
bp product with P1 and P3. (C ) Growth tests of mid-log
cultures, and 1:10 and 1:100 dilutions, spotted on to TAP medium
containing: no selective agent; Spc, 5-FC, and Spc+5-FC. Cultures are
WT, control strain CrCD expressingcodA ,[32] representative transformant lines
generated using plasmid pC-A, and pC-IEE-A.