Fig. 5
From: Split intein-mediated selection of cells containing two plasmids using a single antibiotic
SiMPl based on hygromycin and puromycin. a, f Root mean square fluctuation (RMSF) of Cα atoms in hygromycin B phosphotransferase (PDB ID: 3w0s) (a) and the model structure of puromycin acetyltransferase (f) obtained from protein structure fluctuation simulations via the CABS-flex 2.0 web-server20. Flexible regions, within which splice sites were selected, are indicated by black arrows. b Crystal structure of hygromycin B phosphotransferase (PDB ID: 3w0s). g Tertiary structure model of puromycin acetyltransferase. (b) and (g) Residues that are part of the active site are represented as gray spheres. Splice sites are represented as sticks and are pointed at by black arrows. Section sign (§), splice site that did not support bacterial growth. c, h Bar graph showing the transformation efficiency in E. coli TOP10 cells of the indicated plasmids. Values represent mean (± standard error of the mean) of three independent experiments. d, i Ethidium bromide-stained agarose gel showing plasmid DNA isolated from two clones obtained after transformation with the SiMPl plasmids based on hygromycin (d) and puromycin (i). e Schematic representation of the N-terminal intein construct in pSiMPh_N based on hygromycin. The last few residues of the N-terminal fragment of hygromycin B phosphotransferase are shown. The residue that was mutated in vivo by the bacteria is highlighted (white, WT residue; red, acquired residue). Source data are provided as a Source Data file
