GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules
Figure 4
Multigenic constructs for plant biology.
(A) GoldenBraid cloning path for the assembling of YFP, p19, BFP and DsRED transcriptional units in a single T-DNA. (B) Spatial expression patterns of BFP, YFP and DsRed in N. benthamiana leaves agroinfiltrated with pEGB_A-YFP-P19-BFP-DsRed-C- (left captures, 1, 2 and 3) or with a mixture of the individual devices pEGB_A-YFP-C, pEGB_C-p19-B, pEGB_A-BFP-C and pEGB_C-DsRed-B (right captures 4, 5 and 6). (C) GoldenBraid cloning strategy followed in the assembly of different IgA isotypes. Multipartite assembly involved the combination of different basic parts each occupying a fixed position in the assembly (P1-P5). Individual antibody chains were assembled in pDGB_C12B plasmid to yield four IgA isotypes. 35S is CaMV35S promoter; SP, pectate lyase signal peptide; CHα1 and CHα2, are heavy chain constant domains; Tnos, is nopaline synthase terminator; Cλ and Cκ, are light chain constant domains; VH and VL are heavy and light variable regions of an antibody against rotavirus VP8* peptide. Promoter and terminator pieces were flanked by the same 4 nucleotide extensions as in Fig 1. Signal peptides incorporated a GATG extension at its 5′ end, whereas constant antibody regions ended in TGAG extensions to match terminators. The remaining boundaries were designed to produce benign junctions within coding sequences. (D) Western Blot analysis of IgA transient expression in Nicotiana benthamiana. Leaves were infiltrated with the four previous combinations. Samples were resolved under either reducing (left) or non-reducing (right) conditions and decorated using anti-heavy chain antibody, anti- λ light chain antibody or anti- κ light chain antibody. HS lane contains control human serum. (E) End-point antigen-ELISA tittering of four IgA combinations tested by transient expression in Nicotiana benthamiana leaves. All samples were tittered against VP8* or against BSA and compared with equivalent samples derived from wild type leaves (WT). (F) GoldenBraid strategy for the assembly of two alternative 5-gene T-DNA constructs. (G) PvuI digestion of one colony of each final constructs pDGB_A-KanR-IgHα1-Igλ-Barnase-Rosea-C (lane I) and pDGB_A-KanR-IgHα1-Igλ-Barnase-DsRed-C (lane II). Asterisks highlight those GB-assembled transcriptional units that were reused in the assembly of new multigenic structures.