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Vectors

Vectors. Dr Muhammad Imran. What are plasmids. Are they living, or like viruses? Resistance conferring…..R plasmids Fertility plasmids… F plasmids tra …conjugation Col plasmids bacteriocin …. colicin Degradative plasmids toulene etc Virulence plasmids. E. coli plasmid based vectors.

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Vectors

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  1. Vectors Dr Muhammad Imran

  2. What are plasmids Are they living, or like viruses? • Resistance conferring…..R plasmids • Fertility plasmids… F plasmids tra…conjugation • Col plasmids bacteriocin….colicin • Degradative plasmids toulene etc • Virulence plasmids

  3. E. coli plasmid based vectors • Ease of isolation • High transformation efficiency • Selectable markers for transformants and recombinants • Ability take up large fragments of DNA (up to 8 kb)

  4. Nomenclature • pUC8, pBR322 • P for indicating plasmid based vector • BR for the laboratory they were initially developed in this case Bolivar and Rodriguez laboratory (name of scientist) • 322 is the number of vector…they also developed pBR325, pBR327, pBR328

  5. Salient features of pBR322 • Size < 10 kb • 2 A.B gene • Unique MCS • Insertional inactivation • High copy number 15, 1000-3000

  6. Pedgree of pBR322

  7. Earlier derivative of pBR322 pBR327 • 1089 bp fragment deletion….changed conjugative and replicative ability • Higher copy number 30-45 • Safer to work with….lost ability to conjugate and horizontal transfer of resistance genes

  8. pUC8 …..plasmid ….Univ of California 8 • Derived from pBR322 • No unique restriction sites in AmpR gene • Restriction site clustered in LacZ‘ gene • Much higher copy number 500-700 with out plasmid amplification • Single step selection of recombinant and transformants • Flexibility for cloning MCS is diverse • M13mp shuttling service

  9. Blue White colony selection AmpR Ampicillin resistance IPTG Isopropyl β-D-1-thiogalactopyranoside Xgal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

  10. pGEM3Z—in vitrotranscription of cloned DNA • T7 and SP6 RNA polymerase promoter recognition sequences • 1-2 mg RNA/min • EcoRI, SacI, KpnI, AvaI, SmaI, BamHI, XbaI, SalI, AccI, HincII, PstI, SphI, and HindIIIsites

  11. Phage Cloning vectors • M13 and Lambda phage vectors • Single stranded DNA needed • for sequencing • In vitro mutagenesis GGATTC

  12. Construction of M13 vector

  13. Introducing MCS in M13

  14. Hybrid plasmids-M13 vector • Phagemids………… pEMBL8 1300 bp Helper phage

  15. Construction of Lambda Phage vectors 37-52 is the range 5-25 kb fragment

  16. Deletion 15 kb 18 available

  17. Insertional and replacement vectors • Fragment of lambda is deleted and religated insert can be inserted in this unique site • Either selection with LacZ” or cIinsertional inactivation clear plaques than turbid -------------------------------------------------------------------- • In replacement vector the region between two sites is deleted at the time of cloning and replaced with the right size insert. • Generally on the basis of size

  18. Insertional vectors

  19. Replacement vector 20 kb fragment, size based on Spi phenotype

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