Review Article

Viral Bacterial Artificial Chromosomes: Generation, Mutagenesis, and Removal of Mini-F Sequences

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.
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