Figure 3
Combinatorial assembly of synthetic p-coumaryl-alcohol operons employing Operon-PLICing. Three different variants of each gene, having either a 5, 9 or 13 nt SD sequence–START codon spacing (indicated by different hachures upstream of the respective open reading frames) were generated by PCR with phosphorothioate-oligonucleotides. Subsequently, the phosphorothioate bonds were cleaved for generating complementary 3′-overhangs. The resulting twelve different gene fragments and the vector fragment were mixed in 81 independent hybridization reactions to yield all possible combinations of the tetracistronic p-coumaryl-alcohol operon prior to transformation to E. coli.