Figure 2.

Schematic deletion diagram of TERMP_00517. The plasmid pUPH-1 was constructed by ligation of homologous regions (HR) flanking TERMP_00517 inside pUPH and used to transform T. barophilus MP. After a first homologous recombination (pop-in), cells containing the integrated plasmid were selected with simvastatin (int). The pair of primers 1 was used to verify plasmid integration at this step. Then, intermediated cells were spread on 6-MP to get the second recombination event (pop-out), resulting in plasmid excision. This could lead either to gene deletion or to a WT genotype, depending on the recombination site (full line or dashed line respectively). Primer pairs 2 and 3 are used to validate successful creation of a mutant strain.