Fig 5.

Gene gfp was successfully inserted into the genome of B. multivorans ATCC 17616. (A) Schematic representation of the intergenic region between Bmul_4902 and Bmul_4903 highlighting the place of gfp gene insertion (showed by a green triangle). Letters a/b and c/d show the position of the primers selected to confirm the insertion of the gfp gene before and after plasmid curing, respectively. (B) Electrophoretic separation of the PCR products confirming gfp gene insertion into the genome tested with primers for regions a/b (left panel) and c/d (right panel), this last after plasmid curing. (C) Curing of pCasPA and pACRISPR-Km derivatives using the activity of the sacB gene product. Cells were plated in the shown medium and incubated at 37°C for 24 hours. (D) Fluorescence microscopy (left panel) and light microscopy (right panel) images showing cells of wild-type ATCC 17616 and ATCC 17616 with the gene gfp inserted into chromosome 2.