Figure 2

Multiple site-directed mutagenesis of GFPuv. (a) Three-dimensional structure of GFP (PDB ID: 1GFL) depicted using the ribbon model. Molecular graphics was created with the program UCSF Chimera (http://www.cgl.ucsf.edu/chimera/). The five residues, indicated by yellow balls, were selected as mutation sites because each was essential for GFPuv fluorescence. (b) Wild-type GFPuv in the pBAD vector was used to transform E. coli JM109. The tenth aliquot of each transformant was spread on the right half of an LB agar plate, and the remainder on the left half. The percentage frequency of fluorescent colonies is indicated at the bottom left. (c) A point mutation (Y66D) was introduced in GFPuv by MUPAC. One (d), three (e), or five (f) pre-introduced mutations were simultaneously restored to wild type by MUPAC. CFU in each experiment are listed in Additional file 1: Table S3.