Figure 1.

Assembly of combinatorial expression libraries. (A) Schematic illustration of the MoClo assembly. Basic parts are housed in pUC57-Kan, flanked by BsaI-sites. The T7 promoter is housed in pMA350. The thrombin cleavage site, green fluorescent protein (GFP), one of the proteins of interest (POI), and the terminator (Ter) are assembled in pMA352. The five ribosome binding sites (RBS) and five secretion signals are pooled, and assembled with one of the eight affinity tags (A1–A8) as Pre-libraries in pMA351. The eight affinity tags are: A1, SUMO; A2, His₆-SUMO; A3, MBP; A4, His₆-MBP; A5, GST; A6, His₆-GST; A7, Trx; A8, His₆-Trx. In Level 1, plasmids and inserts are flanked by BbsI sites. For final library assembly, the Pre-libraries are pooled and assembled with the Prefix and Suffix. (B) BsaI digestion of plasmids from a pooled library, and plasmids from single clones. The upper band represents the vector backbone, and the lower bands represent the assembled expression cassettes. Inserts vary in size due to the differing sizes of the affinity tags.