Cloning

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Cloning

• Using Plasmid Vectors

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Vector

• a molecule used as a vehicle to carry foreign
  DNA into a host cell
• Simplest vector plasmid

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Features of Plasmids

• Size
• Functions encoded
• Structure
• Nomenclature
• R-plasmids
• ColE1
• Now standardised

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Why are plasmids suitable cloning vectors?

• Generally do not kill host cell
• Relatively easy to purify
• Can be made small

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Why not use naturally occurring plasmids?

• Too large
• No selectable markers
• Lack of unique recognition sequences for
  restriction enzymes

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Replication

• Plasmid replicon
• Requires an origin of replication (oriV)
• What is required?

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Functions of the ori region

• Host range
• Narrow vs Broad
• Copy number

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Features of Plasmid Cloning Vectors

• Contain an oriV that allows for high copy number,
  may have narrow (pUC) or broad (R) host ranges
• Small why is this an advantage?
• Selectable Genes
• Unique restriction sites
• May have additional features such as mob sites,
  RNA polymerase promoters, etc.

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pBR322

• 1973-1978
• Bolivar and Rodriguez derivative 322
• 4.36 Kb 16 copies per cell
• Narrow host range
• Encodes resistance to ampicillin and to
  tetracycline

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How can we tell if plasmid contains DNA of
interest?

• Insertional inactivation
• Use BamHI site in Tetr gene for cloning
• Transform
• Plate cells on?
• Amp why not Tet?
• Confirm by replica-plating on?
• Tet what do you expect to see?

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Early 1980s pUC series
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Features of pUC Plasmids

• Small
• Very high copy number
• No insertional inactivation

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Blue-White Selection

• Vector contains first 146 aa of the
  b-galactosidase gene (lacZ) (a-peptide)
• MCS embedded within this region

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Blue-White Selection Continued

• Host cell encodes carboxy terminal portion of
  lacZ
• Neither host nor plasmid encodes for entire
  protein
• Together produce enzyme that can cleave Xgal to
  produce blue precipitate

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What if foreign DNA inserted into MCS?

• Foreign DNA will contain a termination codon in
  the same reading frame as the a-peptide
• No a-peptide therefore no b-galactosidase and no
  blue coloured colonies

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pGEM series
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Additional Features

• Origin of DNA replication from ss filamentous
  phage such as F1 or M13
• Phagemid vector
• T7 and SP6 promoters

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www.promega.com
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Variations on the pGEM theme

• pGEMT vectors (Hengen, 1995)
• Make use of unique property of Taq
• Facilitates easy cloning of PCR products - how?
• Limitations?