Figure 1.
Schematic representation of the modular build-up and the overall GB-based cloning strategy of expression vectors.
A) Generic structure of a plastid transformation vector. Magnified details show the modular build-up of a transcriptional unit and how it can be assembled from a set of standardized parts. Numbers within the boxes represent part identity and compatibility. Prom, promoter; Term, terminator; LTR, left targeting region; RTR, right targeting region; UTR, untranslated region; SM, selection marker; GOI, gene of interest; CDS, coding sequence. B) Schematic representation of the cloning strategy yielding the expression vector used in this study. In the pool of standardized parts, circles represent pUPD vectors harboring genetic elements (parts). Elliptical structures represent the different α- and Ω-vectors. Two intertwined ellipses on an arrow represent a one-pot restriction/ligation reaction (GB reaction) combining all the relevant parts. Boxes represent the parts and their assembly.
Figure 2.
A) Schematic representation of the build-up of the expression construct used in this study (not drawn to scale). B) Restriction fragment length polymorphism analysis (RFLP) of the DNA isolated from wild type (WT) plants and two transplastomic lines (1 and 2). C) Northern blot analysis of the aforementioned lines. The lower panel shows total RNA, while the upper represents transcripts labeled with the griffithsin-specific probe. D) Western blot analysis of crude protein extracts of WT plants and two transplastomic lines (1 and 2). The blot was probed with an antibody directed against the hexahistidine tag. + represents an extract of a plant containing thioredoxin with a hexahistidine tag, serving as a positive control.