Deletion of trpA Gene in HV31 Strain

For the deletion of the trpA gene in HV31 (44 ), the strain was transformed with plasmid pTA131-Cupdo (34 (link)), which contains the up- and downstream regions of CRISPR-locus C for integration into the genome. Subsequent selection for loss of the pyrE2 marker by plating on 5-FOA agar led to pop-out colonies. To confirm the removal of trpA, Southern blot analyses were performed. Genomic DNA from potential deletion mutants was digested with SacII, separated on a 0.8% agarose gel, and transferred to a nylon membrane (Hybond^TM-N, GE Healthcare). A 250-bp fragment of the downstream region of locus C was amplified using primers Cdeldoi and DomitteC, radioactively labeled with [α-³²P]dCTP and the DECAprime II DNA labeling kit (Life Technologies) and used as hybridization probe. The resulting strain was termed HV32.