1 / 20

Cloning

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?.

cala
Télécharger la présentation

Cloning

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Cloning Using Plasmid Vectors

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

  3. Features of Plasmids • Size • Functions encoded • Structure • Nomenclature: • R-plasmids • ColE1 • Now standardised

  4. Why are plasmids suitable cloning vectors? • Generally do not kill host cell • Relatively easy to purify • Can be made small

  5. Why not use naturally occurring plasmids? • Too large • No selectable markers • Lack of unique recognition sequences for restriction enzymes

  6. Replication • Plasmid = replicon • Requires an origin of replication (oriV) • What is required?

  7. Functions of the ori region • Host range • Narrow vs Broad • Copy number

  8. 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.

  9. 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

  10. 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?

  11. Early 1980s – pUC series

  12. Features of pUC Plasmids • Small • Very high copy number • No insertional inactivation

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

  14. 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

  15. 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

  16. pGEM series

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

  18. www.promega.com

  19. Variations on the pGEM theme • pGEMT vectors (Hengen, 1995) • Make use of unique property of Taq • Facilitates easy cloning of PCR products - how? • Limitations?

More Related