**1.3** *E. coli* **- An original host and an engineering system for BACs: Size limits for stable cloning**

Of particular import with respect to the direct application of the BACs carrying genes of interest is their modification to render them useful for in-depth reverse genetics research. Some examples on current application and molecular engineering of BAC vectors can be found in other chapters of this book. Currently-available systems for handling large DNA in *E. coli* make it possible to carry out pin-point engineering on DNA carried in BACs. The BAC clone is initially maintained in autonomously-replicating plasmid form in *E. coli*. The prepared BACs are then used for delivery to an *E. coli* BAC-engineering system (Fig. 1b). Genetic manipulations on BACs are summarized in Fig. 2. The maintenance of BACs in *E. coli* requires selection by antibiotics during all stages, as is the case with other plasmid vectors.

Fig. 2. The *B. subtilis* Genome (BGM) system.

Multiple manipulations of large BAC-DNA fragments are possible in BGM; in the *E. coli*  system only single manipulations can be performed. The stability of DNA attributable to the one-copy state of the *B. subtilis* genome assures the maneuverability of *recA-*dependent homologous recombination. Maneuvers can be repeated unlimited times.

The minimal requirements to ensure the stability of BACs before and after engineering operations in *E. coli* are shown in Fig. 2. There are size limitations for the stable handling of BACs in *E. coli;* it has been suggested that the largest clonable size does not exceed 500 kbp. To date not even the existing minimal genome 585-kbp circular one of *Mycoplasma genitalium* has been successfully cloned in *E. coli* (Gibson et al., 2008, 2010)*.*
