Fig. 4

An example of the mechanism behind the generation of genetic modifications in P. thermoglucosidasius using the pMM7 spo0A deletion vector, as constructed from the improved shuttle vector design presented in this study. A Initially, pMM7 is integrated into the genome through homologous recombination at the target site. The successful recombinants are selected by culturing the cells at high temperatures with kanamycin. As the Gram-positive replication of pMM7 is temperature-sensitive, the cells will be forced to integrate the plasmids in order to retain kanamycin resistance. B Due to the presence of sfGFP, successful recombinants will fluoresce green when exposed to blue light. These first recombinants are subcultured without kanamycin for several days, during which some cells will eventually recombine the homologous regions in the vector-originated DNA and the genome for the second time. This results in the loss of the vector DNA. C After the second recombination, the sfGFP has been lost alongside the plasmid DNA, and successful second recombinants can therefore be identified by their lack of fluorescence. The second recombination can produce two outcomes, dependent on which two homologous flanks combined. They can either revert to wildtype or incorporate the intended modification into the strain. In the case of this study, the intended modification was the deletion of sporulation regulator spo0A