FIG. 1.

Map of plasmid pSWEET-bgaB. (a) Significant features of the xylose-based expression system. Plasmid pSWEET-bgaB is a derivative of pDG364 (3), which allows integration into the B. subtilis chromosome at amyE via double recombination and selection with CHL (10 μg/ml). The plasmid also has an E. coli origin of replication, denoted ori, and an ampicillin resistance cassette (50 μg/ml) for routine cloning steps. On the outside of the plasmid map, restriction sites of interest are highlighted, including two PstI sites for convenient plasmid linearization, a PacI site (eight-base recognition sequence TTAATTAA) upstream of bgaB, and a polylinker downstream of bgaB (HindIII is not unique). (b) Close-up of key elements of the xylose expression system (not to scale). Shown are xylR, encoding the xylose repressor; the xyl intergenic region, including promoters for xylR (P_xylR), xylA (P_xylA), and xyl operator sequences (xylO); translationally truncated xylA (first 58 nucleotides followed by an in-frame TAA), including an optimized CRE in xylA (see Materials and Methods and reference 18); PacI 5′ cloning site; ribosome binding site (SD) native to B. subtilis tagD; and gene bgaB, encoding a thermostable β-galactosidase from B. stearothermophilus.