**4. Heavily engineered BACs for downstream pipelines**

Engineered DNA molecules must be delivered to other host systems for diverse applications. The use of BACs for purposes other than sequence determinations, for example, their use in mutagenesis studies on animal models was of limited success (Yang et al., 1997) because complex animal genes range from dozens to hundreds of kbp. They include factors, promoters, 5'untranslated regulatory sequences, introns, exons, splicingand alternative splicing sites, 3'long terminal repeats, and polyA addition sites (Fig. 6b). Functional accessory sequences contain encoding small RNAs and non-coding RNA, and are regulated by, for example, methylation, histone-binding, and nucleosomes. The study of these complex mammal genes by reverse genetic approaches requires systems that facilitate the delivery of larger DNA than is used for microbial reverse genetics where gene units tend not to exceed kbp orders. For such studies, engineered BACs in BGM must be retrievable. A few methods for retrieving DNA from the *B. subtilis* genome are summarized in Fig. 7, three such methods have been applied to retrieve engineered BACs.
