Strategy for carotenoid gene reassembly
The reassembly of the carotenoid genes responsible for synthesising
phytoene, lycopene, and β-carotene was performed as described previously
(Horton et al ., 1995). The sequences of the eight primers used
for reassembly are listed in Supplementary Table S2. Primers ‘a’ and
‘d’, ‘a’ and ‘f’, and ‘a’ and ‘h’ are the flanking primers for PCR
amplification of the final reassembled products. Primers ‘b’ and ‘c’,
‘d’ and ‘e’, and ‘f’ and ‘g’ are the SOE primers. Bases have been added
to the 5′ ends of the primers in each pair to render them complementary.
All of the complementary sequences have been added between primers b‒c,
d‒e, and f‒g. During SOE, the upper strands of AB and the lower strands
of CD overlap to act as primers (Supplementary Fig. S5). Fragment AB was
PCR amplified from crtE , and fragment CD from crtB .
Fragment EF was PCR amplified from crtI , and fragment GH fromcrtY . The SOE primers ’b’ and ’c’ were used to modify the PCR
products of two sequences to have an identical sequence (Supplementary
Table S2). Supplementary Fig. S5B shows the reassembly ofcrtE –B genes for phytoene biosynthesis. The upper strands
of AB and the lower strands of CD overlap to act as primers when the PCR
products are mixed, denatured, and reannealed during PCR. Fragments of
AD are formed when this overlap is extended by polymerase. Supplementary
Fig. S5C shows the crtE –B –I gene reassembly for
lycopene biosynthesis in which the upper strands of AD and the lower
strands of EF overlap to act as primers when the PCR products are mixed,
denatured, and reannealed during PCR. Fragments of AF are formed when
this overlap is extended by polymerase. Similarly, the upper strands of
AF and the lower strands of GH overlap to act as primers when the PCR
products are mixed, denatured, and reannealed during PCR. Fragments of
AH are formed when this overlap is extended by polymerase (Supplementary
Fig. S5D). The XhoI recognition sequence and lacZ RBS were
introduced at the beginning of the SOE-AB products.