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Cloning. Using Plasmid Vectors. Vector. = a molecule used as a vehicle to carry foreign DNA into a host cell Simplest vector = plasmid. Features of Plasmids. Size Functions encoded Structure Nomenclature: R-plasmids ColE1 Now standardised. Why are plasmids suitable cloning vectors?.
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Cloning Using Plasmid Vectors
Vector • = a molecule used as a vehicle to carry foreign DNA into a host cell • Simplest vector = plasmid
Features of Plasmids • Size • Functions encoded • Structure • Nomenclature: • R-plasmids • ColE1 • Now standardised
Why are plasmids suitable cloning vectors? • Generally do not kill host cell • Relatively easy to purify • Can be made small
Why not use naturally occurring plasmids? • Too large • No selectable markers • Lack of unique recognition sequences for restriction enzymes
Replication • Plasmid = replicon • Requires an origin of replication (oriV) • What is required?
Functions of the ori region • Host range • Narrow vs Broad • Copy number
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.
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
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?
Features of pUC Plasmids • Small • Very high copy number • No insertional inactivation
Blue-White Selection • Vector contains first 146 aa of the b-galactosidase gene (lacZ) (a-peptide) • MCS embedded within this region
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
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
Additional Features • Origin of DNA replication from ss filamentous phage such as F1 or M13 • Phagemid vector • T7 and SP6 promoters
Variations on the pGEM theme • pGEMT vectors (Hengen, 1995) • Make use of unique property of Taq • Facilitates easy cloning of PCR products - how? • Limitations?