Bacterial Artificial Chromosome Libraries Jonathan Garcia

Cloning Bacterial Artificial Chromosomes(BAC) utilize a cloning system derived from a plasmid found in Ecoli. Allows one to develop much larger pieces of DNA compared to standard plasmids (10,000bp vs 350,000bp) Reduces numbers of clones need to sequence a genome by 35x First used by Hiroaki Shizuya in 1992

Components in a bac RepE: for plasmid replication and regulation of copy number Hind III and Bam HI: Sites of Cloning oriS: The origin of replication CmR: Chloramphenicol resistance gene, selection tool ParA,ParB,ParC: the genes that regulate the partitioning of plasmids to daughter cells during division.

Mechanism Developed by taking parts of of the F’ and turning it into a vector. The origin of replication allows the host cell to recognize the new DNA insert. Location of HindiII and BAmHI within LacZ changes X-gal metabolism when target DNS is incorporated CmR induces antibiotic resistance to target DNA containing vectors

Vector and donor DNA both cut by same restriction enzyme Fragments are mixed and sticky ends hybridize via H-bonds DNA Ligase seals gaps by forming phosphodiester linages

Method DNA Fragments of interest are isolated and cleaved using restriction enzymes The BAC is digested by restriction enzymes around the cloning site (HindlII and BamHI) Recombinant DNA is formed (F’plasmid and target DNA) using DNA ligase. New recombinant DNA is inserted into compliant cells and plated As bacterial cells grow and divide they also amplify the BAC DNA which can be isolated CmR and LacZ distinguish between successful transmission of target gene into bacterium.

Modifications Modifications can be made to make the BAC vectors more specialized sacB encodes a protein called levansucrase that turns sucrose into levan, a toxic substance to bacteria, so when unbroken will cause cell death. Insertion of entire Viral genomes pSV1.RecA, which possesses a recombinase gene and a temperature-sensitive origin of replication

Applications BACs allow cloning and maintenance of large segments of DNA, making them useful in whole genome mapping Independent Chromosome allows easy isolation, and pure inserts. Allows genetic information to be stored from organisms that aren’t easily grown in cultures. Study of large pathogenic viruses

References W.C. Nierman, T.V. Feldblyum, in Encyclopedia of Genetics, 2001 G.M. Weinstock, in Encyclopedia of Genetics, 2001 “What Are BAC Libraries?” Facts, The Public Engagement Team at the Wellcome Genome Campus, 25 Feb. 2015, www.yourgenome.org/facts/what-are-bac-libraries. StudiousGuy. “Cloning Vectors: Types & Characteristics.” StudiousGuy, StudiousGuy, 19 Sept. 2018, studiousguy.com/cloning-vectors-types-characteristics/. Kevinshe. “THE BIG BAD BAC: BACTERIAL ARTIFICIAL CHROMOSOMES.” SCQ, 6 Sept. 2006, www.scq.ubc.ca/the-big-bad-bac-bacterial-artificial-chromosomes/.

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