**6. Future perspectives**

132 Bacterial Artificial Chromosomes

Accommodation in\*3 BGM by Fig.1b

F1O22 101 **Yes Yes Yes** F2L14 100 **Yes Yes Yes** F3A21 100 **Yes Yes Yes** F3B3 115 **Yes Yes Yes** F4C19 100 **Yes Yes Yes** F4I8 100 **Yes Yes Yes** F4O20 144 **Yes Yes Yes** F6A21 115 **Yes Yes Yes** F6A7 105 **Yes Yes Yes** F7I2 85 **Yes Yes Yes** F7J2 98.5 **Yes Yes Yes** F9J20 120 **Yes Yes Yes** F10J8 90 **Yes Yes Yes** F10L13 89.5 **Yes Yes Yes** F10F17 100 **Yes Yes Yes** F11E12 80 **Yes Yes Yes** F13E8 90 **Yes Yes Yes** F13O24 115 **Yes Yes Yes**

pKANED†2 150 **Yes (**NT) (NT)

 110 **Yes (**NT) (NT) 86 **Yes (**NT) (NT) 34 **Yes Yes Yes** 16.5 **Yes Yes (**NT) 11.2 **Yes Yes Yes**

pKANEG†3 198 **Yes** (NT) (NT) pKANEH†3 219 **Yes** (NT) (NT)

p185A21 120 **Yes** (NT) (NT) \*1BAC clones in literature \*2Estimated by gel electrophoresis. \*3Confirmed by Southern blot analysis using original BAC clone as probes. \*4Used for transformation of *E. coli* strains DH1 or DH10B. Refereed

by †1 Kaneko et al., 2005, †2 Kaneko et al., 2003 †3 Kaneko et al., 2008, and †4 Itaya et al., 2000.

355 **Yes** (NT) (NT)

(Deletion of pKANED as in Fig.5a and 5b)

Retrieval in plasmid as Fig.7

Restored in\*4 *E. coli*

DNA source from

insert\*2 size (kbp)

*Arabidopsis thaliana* **mtDNA**†1

**Mouse** *Jmj* **gene (Chromosome 13)** 

(Connection: pKANEG +pKANEH in Fig.6a)

Table 1. BAC clones\*1 handled by BGM systems

**Mouse** *TS* **gene DNA (Chromosome 11)** †4

Based on available data, we think that the use of *B. subtilis* will facilitate the rapid and efficient engineering of BACs. While both *E. coli* and *B. subtilis* are excellent BAC hosts, several experimental steps require that DNA be manipulated outside these hosts. Steps involved in the delivery of DNA from *E. coli* to BGM and in the isolation of engineered guest DNA render the fragments maintained in liquid vulnerable to damage due to the intrinsic physicochemical nature of these liquids.

Elsewhere we reported that the conventional delivery step may be replaced (Kaneko & Itaya, 2010a, 2010b; Itaya & Kaneko, 2010). We fortuitously found that plasmid DNA remains intact for a while after the induction of lambda phage of *E. coli*. While *E. coli* was lysed and its genome degraded, the co-existing plasmid remained intact and available for transfer to competent *B. subtilis*. The stability of plasmids in the lambda-induced *E. coli* lysate also applied to BACs (Kaneko & Itaya, 2010a, 2010b; Itaya & Kaneko, 2010)**.** Consequently, it is no longer necessary to attempt to prepare undamaged BACs in test tubes.

The techniques involved in the isolation of undamaged DNA from electrophoresis gels have been improved and applied to fragments of DNA measuring up to 220 kbp. In the postgenome-sequencing era, reverse genetics using designed and manipulated BACs and BGM will play an essential role in the construction of various genetic mutants.
