Introduction
Bacterial artificial chromosomes (BACs) are single-copy-number, high-capacity circular DNA vectors derived from Escherichia coli fertility factor (F-plasmids). BACs are used for transforming and cloning in bacteria, accommodating approximately 150 – 350 kb of inserted DNA. The BAC system utilizes three key F-factor components: (1) the origin of replication (oriS) and repE gene, (also known as RepA) mediates assembly of a replication complex at OriS (LeBowitz & McMacken, 1984). (2) partition genes parA and parB, which maintain the characteristic 1-2 copy number per cell. (3) parB, which excludes extraneous F plasmids from cells. These features ensure equal segregation during cell division and stable maintenance of BACs in daughter cells.
The first BAC vector, pBAC108L, developed through deletion of unnecessary F-factor plasmid sequences, and addition of antibiotic resistance maker (camR – chloramphenicol). This vector enabled cloning and stable maintenance of DNA inserts up to 300 kb in E. coli. Subsequently, pBeloBAC11 constructed by incorporating the lacZ gene to provide selectable markers for recombinant clones through blue–white screening. This article focuses on pBeloBAC11.
pBeloBAC11
pBeloBAC11, a F-plasmid-derived vector engineered for cloning large DNA fragments in E. coli strain DH10B [4]. The vector maintains single-copy status per cell and can accommodate inserts up to 300 kb with remarkable stability and faithful replication. Its incorporation of the lacZ reporter gene facilitates easy identification of recombinant clones, making it valuable for genomic studies and DNA library construction. [2]
Similar to pBAC108L, the cloning segment of pBeloBAC11 contains several essential elements: (1) a bacteriophage λ cos site (cosN) and a loxP site. The cosN site required for packaging DNA into λ phage particles, while both sites are crucial for restriction mapping. The λ cosN site provides a fixed position for specific cleavage by λ terminase, while the loxP site enables Cre recombinase-mediated recombination. (2) Two unique cloning sites, HindIII and BamHI, flanked by T7 and SP6 promoters. Promoters facilitate the generation of RNA probes for sequencing and bloting procedures, and probably chromosome walking. (3) GC-rich restriction sites (e.g., NotI, SmaI, SfiI) flanking the cloning site enable potential excision of inserts.
Construction of BAC libraries
Digest pBeloBAC11 with BamHI and dephosphorylate to prevent self-ligation. Partially digest target genomic DNA with BamHI and size-select fragments using pulsed-field gel electrophoresis. Combine prepared vector and size-selected fragments. Electroporate ligated products into E. coli DH10B Selection: Plate transformants on media containing chloramphenicol, X-gal, and IPTG; identify recombinants as white colonies via blue-white screening
Application of BACs
Advantages of BACs
Bacterial artificial chromosomes (BACs) address key limitations of yeast artificial chromosomes (YACs). Unlike YACs, which are prone to chimerism, instability, and challenging DNA isolation, BACs maintain large genomic DNA fragments (>300 kb) as stable, single-copy supercoiled plasmids in recombination-deficient E. coli hosts. This system offers simplified library construction and improved insert stability.
pBeloBAC11 serves as a crucial tool in genomic research with three primary applications: (1) construction of genomic DNA libraries for complex organisms, (2) high-throughput genome sequencing projects requiring stable large-insert clones e.g, for example it was used during Human Genome Project and (3) functional genomic studies, including positional cloning and physical mapping of genes.
BACs can also be utilized to detect genes or large sequences of interest and then used to map them onto the human chromosome using BAC arrays[5].
Limitation of BACs
Although the construction of BAC libraries is relatively easy, several steps can be tedious. The single-copy nature of the vector pBeloBAC11 makes purification of this plasmid laborious [4].
Difference between YAC and BAC Vectors
The key differences between YAC (Yeast Artificial Chromosome) and BAC (Bacterial Artificial Chromosome) vectors.
References
- LeBowitz, J. H., & McMacken, R. (1984). The bacteriophage λ O and P protein initiators promote the replication of single-stranded DNA. Nucleic Acids Research, 12(7), 3069–3088. https://doi.org/10.1093/nar/12.7.3069.
- Ávila-Pérez G, Park JG, Nogales A, Almazán F, Martínez-Sobrido L. Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone. J Vis Exp. 2019 Jun 24;(148).
- Daniel Tillett & Brett A. Neilan (1998) Small-Scale Preparation of the Single
- Copy Bacterial Artificial Chromosome Vector pBeloBAC11, BioTechniques, 24:4, 568-572, DOI:10.2144/98244bm10.
- Bajpai, B. (2013). High capacity vectors. In Springer eBooks (pp. 1–10). https://doi.org/10.1007/978-81-322-1554-7_1.
- Reece, R. J. (2004). Analysis of genes and genomes. Wiley.
- Full-length Human cDNA Clone Collection
