Fig. 1.

Creation and screening of a xylose-stimulated xylanase chimera. a The pT7T3GFP_XBP plasmid containing the xylose inducible GFP gene and XBP gene (xylF) under a constitutive promoter was randomly linearized and ligated to a xylanase gene (xynA from B. subtilis), resulting in a library of random insertions of xylanase into this plasmid. b After transformation of Escherichia coli with genomic XBP knocked out, the library was screened for functional XBP using a gene circuit for increased fluorescence in the presence of xylose. The XBP+ clones were separated by cell sorting and were plated on selective TB-agar media, supplemented with xylan in the presence of xylose, and the clones expressing xylanase activity (XynA+) were identified by the formation of halos on solid agar xylan plates after staining with Congo red. Xylose-stimulated catalytic activity in supernatants of XBP+/XynA+ clones were assayed for hydrolysis of RBB-xylan in the presence and absence of xylose