**3.2 NCI Frederick bacterial based system**

The National Cancer Institute http:// web.ncifcrf.gov/ research/ brb/ recombineering Information.aspx offers E. coli strains that contain the phage recombination proteins stably integrated into the genome. The proteins are under transcriptional control of the λPl promoter in concert with the temperature sensitive cl857 repressor. Transcription of the lambda phage proteins is repressed at 32C. Repression of the λ Pl promoter is released by incubating cells at 42C for 15 minutes. Placing the stably integrated phage proteins under tight transcriptional control circumvents the problems of unwanted recombination associated with constitutive expression (Warming, Costantino et al. 2005). NCI offers strains with an L-arabinose inducible Cre or Flpe expression, which are useful for excision of the selectable antibiotic markers used for positive selection of recombinants.

In addition, NCI has developed plasmids to facilitate the cloning of sequence from BAC DNA into high copy plasmids for downstream use as targeting vectors (Liu, Jenkins et al. 2003). Plasmids for epitope tagging new proteins of interest have also been developed, allowing the investigator to localize and purify proteins. This application speeds the functional characterization of proteins by eliminating the lengthy process of generating antibodies that would serve similar functions (Poser, Sarov et al. 2008).
