Figure 1: Schematic illustration of shuttle mutagenesis. In a first step, a shuttle plasmid is inserted into the target sequence via RecA-mediated recombination of homologous sequences. Replication of the shuttle plasmid containing a temperature sensitive origin (oriTS) is repressed by a temperature increase to 42°C. Positive co-integrates are selected with corresponding antibiotics. In a second step, vector sequences are excised from co-integrates by another recombination. Negative selection markers can be used to select BAC constructs that lost the shuttle plasmid [28, 64]. Dotted lines symbolize recombination events.