Bacterial artificial chromosome (bac)

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Mel Simon and Co-workers developed the BAC which is another cloning vector system in E. coli and an alternative to YAC vector vectors. BACs are maintained in E. coli as a large single copy. These are used as vectors for cloning very large (>50 kb) sequences of DNA. Some times these fragments turn out to comprise non-contiguous (non-adjacent) segments of the genome and frequently lose parts of the DNA during propagation. Hence, these become unstable.
BAC are constructed by using fertility or F factors (present on F plasmid) of E.coli. BAC vectors contain ori gene, repE gene for maintenance of F factor, genes (parA, B, C) for plasmid copy number, an antibiotic resistance gene e.g. chloram-phenicol acetyltransferase (chlR) gene for selection of plasmid and many restriction sites for insertion of foreign DNA. The F factor encodes its own DNA polymerase and is maintained in the cell as one or two copies. BACs are maintained as single copy plasmids in E. coli excluding the replication of more than one BAC in the same host cell. The upper limit of foreign DNA to be inserted in BAC is about 300-3500 kb. BACs are alternative to YACs and, therefore, being used in geneome sequencing projects.
The method of preparation of BAC.library is the same as for plasmid library. But here the DNA insert is prepared by pulse field gel electrophoresis (PFGE). PFGE has made it possible to separate, map and analyse very large DNA fragments.
Large sized globular DNA migrates easily after applying discontinuous electric field. Example of a BAC vector is pBeloBAC 11(Fig.) which is partially digested while cloning a DNA fragment. Genomic DNA is partially digested with HindIII. Size fractionation of DNA is made by PFGE. Similarly, pBeloBAC II is treated with HindIII and phosphatase. Such digested vector and fragments of genomic DNA (100-300 kb) are ligated.
The recombinant plasmid containing DNA insert is transferred into E. coli cells through electroporation. E. coli can be transformed very efficiently by BAC. Then E. coli cells are plated on LB medium containing chloramphenicol, IPTG and X-gal for selection. White colonies of E. coli are picked up based on chloramphenicol resistance.
In 1989, by using this vector DNA segments from bithorax gene of Drosophila have already been cloned. BAC clones are stable for many generations. In contrast, it lacks positive selection for clones that contain DNA inserts. Therefore, the yield of DNA is very low.

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