**4.3 Retrieval by copying segments of the** *B. subtilis* **genome**

The third method involves a yet more complicated genetic process referred to as *Bacillus* Recombinational Transfer (BReT) (Tsuge & Itaya, 2001; Kaneko et al., 2005). Indeed, BReT relies on homologous recombination in *B. subtilis* and should be as simple as the genetic disconnection procedure described above. Different from the disconnection procedure, the engineered BAC parts from the genome are copied and pasted into the existing plasmid. DNA retrieval from the genome to the plasmid involves a reverse direction of the DNA delivery into a host pBR322 or BAC as illustrated in Fig. 1b. The complete circular form yielded by the BReT pathway, selection with plasmid-linked markers followed by standard extraction of plasmid DNA, resulted in the purification of complete recombinant genomes of lambda (Tsuge & Itaya, 2001), organelle genomes from mitochondria and chloroplast (Itaya et al., 2008; Yonemura et al., 2007), and several BACs (Kaneko et al., 2005). The engineering of organelle genomes, mitochondria, and chloroplasts opens an exciting field because typical organelle genome sizes, ranging from 100-200 kbp for chloroplast, are below the carrying capability of BACs. Although there are currently no reliable technical tools to deliver them back to natural cells, the availability of circular freely engineered mitochondria or chloroplast genomes raises the need for such tools (Gibson et al., 2008, 2010; Itaya et al., 2008; Itaya, 2010).
