Bacterial artificial chromosomes

Ideal BioBricks single copy vector

• F plasmid backbone
• ccdB and pUC19 backbone in between BioBricks restrictions sites
• strong terminators flanking the BioBricks insertion site
• no loxP or cos insertion sites or Tn7 attachment sites
• multiple versions with different antibiotic resistance markers
• no selection system for mammalian cells
• VF2 and VR sites

Existing vectors

BAC vectors

• Circular DNA molecules which contain a replicon that is based on the F factor. It has oriS and repE which encode an ATP-driven helicase and parA, parB and parC for partitioning.
• original BAC vectors are 7.4 kb, others are 8-9kb.
• can clone 80-300kb fragments, sometimes more
• usually the host need to be deficient in homologous recombination machinery in order for the BAC to be stable (i.e. recA^-). Usually DH10B is used as the host.
• can retrofit BAC vectors with a particular insert by using a retrofitting vector like pRetroES (Ref 3)

pBAC108L

• no selection system for inserts
• one of the original vectors
• H. Shizuya, B. Birren, U. J. Kim, V. Mancino, T. Slepak, Y. Tachiiri, and M. Simon. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci USA, 89(0027-8424):8794–7, 1992.

pBeloBAC11

• recommended by Tom, have this in the lab
• screen for inserts via α-complementation (blue-white screening on IPTG/Xgal plates)
• does not have a selection marker for transfection into mammalian cells
• pBeloBAC11 from NEB
• cos site
• loxP site
• inserts up to 1Mb
• T7 and SP6 promoters flank the insertion site

pBACe3.6

• transferred the pUC-link and SacBII sequences from a PAC vector to pBAC108L. See PAC vectors below.
• does not have a selection marker for transfection into mammalian cells
• pBACe3.6 information/map
• E. Frengen, D. Weichenhan, B. Zhao, K. Osoegawa, M. van Geel, and P. J. de Jong. A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites. Genomics, 58(0888-7543):250–3, 1999. pubmed
• Genbank accession number U80929
• has a loxP site for Cre recombinase protein
• 11.5 kb
• has lots of BioBricks sites internal to the vector backbone

pFos1

• pFos1 from NEB
• "Grow cells at 30°C. The plasmid is unstable at 37°C."
• "The plasmid pFOS1 carries a gene encoding resistance to ampicillin (amp) and a gene encoding resistance to chloramphenicol (cam). To maintain the plasmid, cells should be grown with 100 μg/ml amp and 15-20 μg/ml cam."
• U. J. Kim, H. Shizuya, P. J. de Jong, B. Birren, and M. I. Simon. Stable propagation of cosmid sized human DNA inserts in an F factor based vector. Nucleic Acids Res, 20(0305-1048):1083–5, 1992.
• "constructed by fusing pBAC (Shizuya et al., in preparation), an F replicon based plasmid with lambda cosN site, to pUCcos, a pUC derivative containing cosN site, by homologous recombination in E. coli through the shared cosN site. pBAC and pUCcos were transformed to the strain C600r-m+ sequentially, and the transformants grown on LB plates containing ampicillin and chloramphenicol were picked. Miniprep plasmid DNA prepared from the transformant containing fused replicons was diluted and transformed to the strain D 1OpolA-, in which pUC replication origin cannot function and therefore only the plasmids containing the F factor replication origin can survive.
• thus for this vector to be at single copy, it must be in a strain in which pUC origin is inactive

pFW11 and F' factor

• F. W. Whipple. Genetic analysis of prokaryotic and eukaryotic dna-binding proteins in escherichia coli. Nucleic Acids Res, 26(0305-1048):3700–6, 1998. pubmed (from Natalie)
• available through Natalie from an HMS lab
• clone insert into pFW11 between 3' end of lacI and 5' end of lacZ. Transferred via homologous recombination to the complete lac operon (lacZYA) on the F' episome. Then F' moves to another strain via conjugation which is distinguished from the original strain by streptomycin resistance.
• distinguish between single recombinants (both episomes) and double recombinant F' via the marker sensitivity.
• method designed to test DNA binding proteins and cognate promoters via β-galatosidase activity.
• pFW11 has a strong terminator upstream of the polylinker
• may be a more sophisticated system than we need?

pCC1BAC

• CopyControl pCC1BAC vector from Epicentre
• "contains the E. coli F-factor single-copy origin of replication and an inducible high-copy oriV origin of replication."
• "grown at single-copy number to ensure insert stability. Then, clones can be induced to 10-20 copies per cell within 2 hours of adding CopyControl Induction Solution to the culture, for higher yields and higher purity DNA. Although large-insert clones are less stable when cloned and maintained in high-copy vectors, the short 2-hour induction of CopyControl BAC clones to high-copy-number does not decrease their stability, based on analysis of Hind III restriction patterns of a large number of induced versus uninduced clones that ranged in size up to ~200 kb."

pIndigoBAC-5

• pIndigoBAC-5 from Epicentre
• "derived from pBeloBAC11 and pIndigoBAC1 and will accommodate and stably maintain DNA inserts of >100 kb."
• "linearized at its unique BamH I or Hind III site, dephosphorylated, and highly purified and is ready for cloning BamH I- or Hind III-cut genomic DNA. The linearized and dephosphorylated vectors are tested to ensure the completeness of linearization, dephosphorylation, and the integrity of the BamH I and Hind III ends."
• "The complete sequence and restriction map of pIndigoBAC-5 is available."
• "Enhanced blue/white screening of recombinants."

Cosmids

• Cosmids: conventional vectors that contain a cohesive end site (cos) from bacteriophage λ

pWEB-TNC^TM

• pWEB-TNC from Epicentre
• "The kits utilize a novel strategy of cloning end-repaired, randomly sheared DNA instead of the conventional approach of cloning fragments generated by partial restriction endonuclease digestion. First, genomic DNA is sheared by passing it through a syringe needle. The sheared DNA is end-repaired to generate 5’-phosphorylated blunt ends and size-selected using a low melting point agarose gel. The size-selected DNA is then ligated into the supplied linearized and dephosphorylated pWEB-TNC^TM or pWEB^TM Cosmid Vector, packaged using ultra-high efficiency MaxPlax^TM Lambda Packaging Extracts (>109 pfu/μg for phage lambda) and plated on phage T1-resistant EPI100^TM-T1R E. coli plating cells, all included in the kit. The result is a complete and unbiased primary cosmid library."
• Has EcoRI, NotI ... NotI, EcoRI in the multiple cloning site making it fairly easy to create a BioBricks insertion site in the plasmid.
• Has a ColEI origin annotated on the vector.

SuperCos 1

• SuperCos 1 from Stratagene
• we have it?
• Tom says we got it from Epicentre
• "Cosmid vectors are valuable tools in this analysis, because they can accommodate genomic DNA fragments ranging in size from 30 to 42 kb."
• "SuperCos 1 is a novel, 7.9-kb cosmid vector that contains bacteriophage promoter sequences flanking a unique cloning site."
• "The SuperCos 1 vector is also engineered to contain genes for the amplification and expression of cosmid clones in eukaryotic cells."
• "Most genomic inserts can be excised as a single large restriction fragment using the Not I restriction site that flanks the SuperCos 1 polylinker."
• cos sites are for in vitro packaging with λ
• has a pUC origin on the map.

PAC vectors

• derived from bacteriophage P1 vector
• 15-16kb
• positive selection for inserts via sacB the levansucrase gene which converts sucrose to levan which is toxic to E. coli
• a pUC-vector-derived fragment is inserted into the cloning site which inactivates sacB and allows large amounts of vector to be purified. Your insert replaces this pUC vector thereby returning the vector to single copy. Must be careful to ensure that no uncut vector is present in cloning because it will transform as well as the insert containing vectors. Thus, the sacB marker only prevents against vector religation productions not intact vector.

Protocol notes

BAC purification

• exist as supercoiled circular plasmids and are resistant to shearing making purification easier
• most purification protocols appear to be geared for preparation of genomic libraries for sequencing. It is not clear if we need such elaborate protocols for simple clonings.
• Typical DNA yields
  1. 1μg from 5mL LB culture sing alkaline BAC DNA purification
  2. 0.8μg from 1.3mL TB culture using Qiagen R.E.A.L.
  3. 4μg from 20mL LB culture using Qiagen-tip 20
  4. 100μg from 500mL LB culture using Qiagen-tip 500

Putting the BAC into E. coli

1. Transformation
   • has low efficiencies
   • works on any size
2. In vitro packaging
   • package the vector inside λ bacteriophage particles and infect cells
   • limits the size of the DNA because of phage head stability. The phage head is not stable if the vector is too small or too large. Usually ~40kb (between 25-45 kb okay) insert with an 8-9kb vector works.
   • upon introduction into the E. coli cells the vector is circularized to for a large plasmid with the insert.

F factors and F factor derived vectors

• Biol301 at Eastern Michigan University

Nomenclature

• Episome: A circular piece of DNA that can replicate independently of the bacterial chromosome or integrate and replicate as part of the chromosome.
• F factor: An episome in bacterial cells that confers the ability to act as a genetic donor during conjugation.
• F+ cell: A bacterial cell possessing the fertility factor. Acts as a donor in conjugation.
• F- cell: A bacterial cell that does not contain a fertility factor. Acts as a recipient in conjugation.
• F' cell: A bacterial cell in which an integrated fertility factor spontaneously excises itself, taking part of the chromosome with it. The resulting episome therefore contains chromosomal material.
• Hfr cell: A strain of bacteria exhibiting a high frequency of recombination, due to a chromosomally-integrated fertility factor. This strain is able to transfer all or part of the chromosome to recipient strains.

Mating types

• F+ mating with F- produces 2 F+
• Hfr mating with F- produces Hfr and F-
• F' mating with F- produces F' and F' merozygote

References

1. F. W. Whipple. Genetic analysis of prokaryotic and eukaryotic dna-binding proteins in escherichia coli. Nucleic Acids Res, 26(0305-1048):3700–6, 1998. pubmed (from Natalie)
2. Bacterial artificial chromosomes. S. Zhao and M. Stodolsky, editors, Volume 1: Library Construction, Physical Mapping, and Sequencing, volume 255 of Methods in Molecular Biology. Humana Press, 2004. (from Tom)
3. Bacterial artificial chromosomes. S. Zhao and M. Stodolsky, editors, Volume 2: Functional Studies, volume 256 of Methods in Molecular Biology. Humana Press, 2004. (from Tom)
4. Evans, G. A., Lewis, K. and Rothenberg, B. E. (1989). High efficiency vectors for cosmid microcloning and genomic analysis. Gene 79: 9-20.
5. Hohn, B. and Collins, J. (1980). A small cosmid for efficient cloning of large DNA fragments. Gene 11: 291-298.
6. E. Frengen, D. Weichenhan, B. Zhao, K. Osoegawa, M. van Geel, and P. J. de Jong. A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites. Genomics, 58(0888-7543):250–3, 1999. pubmed
7. Biol301 at Eastern Michigan University
8. E. coli plasmid vectors. N. Casali and A. Preston, editors, Methods and Applications, volume 235 of Methods in Molecular Biology. Humana Press, 2003.

• H. Shizuya, B. Birren, U. J. Kim, V. Mancino, T. Slepak, Y. Tachiiri, and M. Simon. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci USA, 89(0027-8424):8794–7, 1992.

1. U. J. Kim, H. Shizuya, P. J. de Jong, B. Birren, and M. I. Simon. Stable propagation of cosmid sized human DNA inserts in an F factor based vector. Nucleic Acids Res, 20(0305-1048):1083–5, 1992.