ArticleGenome research2002
Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones.
Article in Genome research, 2002. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 80 papers.
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Who cites it
80 citing papers in PubMed.
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- Genomic context sensitizes regulatory elements to genetic disruption.Molecular cell · 2024Article
- Genomic context sensitizes regulatory elements to genetic disruption.bioRxiv : the preprint server for biology · 2024Article
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- Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus.Molecular cell · 2023Article
- Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster.Current protocols · 2023Article
- Functional Metagenomics as a Tool to Tap into Natural Diversity of Valuable Biotechnological Compounds.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Reverse genetics systems for SARS-CoV-2.Journal of medical virology · 2022Review
- Construction and characterization of two SARS-CoV-2 minigenome replicon systems.Journal of medical virology · 2022Article
- Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct.Molecular therapy. Methods & clinical development · 2020Article
- GPAHex-A synthetic biology platform for Type IV-V glycopeptide antibiotic production and discovery.Nature communications · 2020Article
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- Discovery of Novel Biosynthetic Gene Cluster Diversity From a Soil Metagenomic Library.Frontiers in microbiology · 2020Article
- A Fosmid-Based System for the Generation of Recombinant Cercopithecine Alphaherpesvirus 2 Encoding Reporter Genes.Viruses · 2019Article
- Genetic Engineering by DNA Recombineering.Current protocols in chemical biology · 2019Article
- Plasmid-based gap-repair recombineered transgenes reveal a central role for introns in mutually exclusive alternative splicing in Down Syndrome Cell Adhesion Molecule exon 4.Nucleic acids research · 2019Article
- Molecular Insights into Antimicrobial Resistance Traits of Commensal Human Gut Microbiota.Microbial ecology · 2019Article
20 more citing papers are in PubMed but not listed here.
Corrections and comments
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Authors and funding
3 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome
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