Bacterial artificial chromosomes

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Contents

Existing vectors

BAC vectors

  • F-factor plasmid based vectors
  • 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

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

pBACe3.6

  • transferred the pUC-link and SacBII sequences from a PAC vector to pBAC108L. See below.
  • does not have a selection marker for transfection into mammalian cells

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 insert with an 8-9kb vector works.

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.
  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.
  3. Bacterial artificial chromosomes. S. Zhao and M. Stodolsky, editors, Volume 2: Functional Studies, volume 256 of Methods in Molecular Biology. Humana Press, 2004.
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