**3.2 Direct manipulation of guest BACs**

As guest DNA replicates as part of the host genome, sequence modifications/conversions targeting regions in the *B. subtilis* genome are possible (Fig. 4). In addition to sequence conversions or small DNA insertions, the formation of systematic deletions was examined for a guest DNA derived from the mouse genome. The protocol to induce deletion shown in Fig. 5a applies a method that is the reverse of the method for integration; it uses two homologous recombinations. Two small DNA segments with deletion endpoints are connected so as to flank appropriate antibiotic resistance markers for *B. subtilis*. The region to be deleted was replaced by a marker gene via two homologous recombinations at the two flanking segments. Kaneko et al. (2003) succeeded in inducing four deletions ranging from 11 to 86 kbp in the mouse *jumonji* (*jmj*) gene locus (Fig. 5b). The preparation of DNA tools by using conventional gene engineering technologies in *E. coli* plasmids and their introduction into competent BGM are now routine. We do not think that such designed and wellcontrolled deletion formation is possible using standard BAC manipulation kits developed for *E. coli*. Other auxiliary tools, antibiotics resistance genes, and rare-cutting endonucleases, to apply and improve these protocols are presented in Chapter 4 below.

Fig. 5a. Concept underlying the formation of deletions.

Transformation using the optional small DNA fragments and antibiotic resistance markers for *B. subtilis* produces the designed deletion formation via homologous recombination.

Fig. 5b. Systematic deletion formation from a mouse genomic region (110kb). The BGM system makes it possible to conduct massive and systematic deletions. The picture on the right includes I-*Ppo*I fragments resolved by gel electrophoresis (open arrowheads with the size of the deletion indicated on the left). The BGM vector (4.2 Mb) migrates slowly.

Transformation using the optional small DNA fragments and antibiotic resistance markers for *B. subtilis* produces the designed deletion formation via homologous recombination.

Fig. 5b. Systematic deletion formation from a mouse genomic region (110kb).

The BGM system makes it possible to conduct massive and systematic deletions. The picture on the right includes I-*Ppo*I fragments resolved by gel electrophoresis (open arrowheads with the size of the deletion indicated on the left). The BGM vector (4.2 Mb) migrates slowly.

Fig. 5a. Concept underlying the formation of deletions.
