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Bacterial Artificial Chromosome Libraries

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)

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Bacterial Artificial Chromosome Libraries

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  1. Bacterial Artificial Chromosome Libraries Jonathan Garcia

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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/.

  10. Questions

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