Figure 4: (A ) Illustration of the plastome loci (i.e.
downstream of psbH or psbA ) prior to transgene insertion
(WT), as the intermediate recombinant loci (R1), and following loop-out
of the codA-aadA marker (R2). The locations of the primers (P1 –
P4) used in the four-primer PCR analysis of transformant lines are
indicated together with the expected product sizes. (B ) PCR
analysis of six transformant lines for each locus indicate that all are
homoplasmic for the R1 plastome following three rounds of selection on
Spc. The WT band is the product of P1+P2, transformants bands are the
product of P3+P2. (C,D ) PCR analysis following two further
rounds of restreaking on TAP or TAP+5-FC media demonstrates the
stability of the R1 genotype in the absence of selection (P3+P2 product
retained), and rapid loss of the codA-aadA cassette under 5-FC
selection (new band corresponding to P4+P2 product). (E )
Luminescence analysis of mid-log cultures of Luc1.R1, Luc1.R2, Luc2.R1
and Luc2.R2 lines. Luminescence units were normalised to optical cell
density (OD750) with values based on 3n replicates.
(F ) Spot tests of mid-log cultures and 1:10 and 1:100 dilutions
of WT and transformant lines C-A, Luc1.R2 and Luc1.R2 on TAP plates
containing: no antibiotic, Spc or 5-FC.