VectorsDr Muhammad Imran

What are plasmidsAre they living, or like viruses?

1. Resistance conferring…..R plasmids2. Fertility plasmids… F plasmids tra…

conjugation3. Col plasmids bacteriocin….colicin4. Degradative plasmids toulene etc5. Virulence plasmids

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)

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

Salient features of pBR322Size < 10 kb2 A.B geneUnique MCSInsertional inactivationHigh copy number 15, 1000-3000

Pedgree of pBR322

Earlier derivative of pBR322

pBR3271089 bp fragment deletion….changed

conjugative and replicative abilityHigher copy number 30-45Safer to work with….lost ability to

conjugate and horizontal transfer of resistance genes

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

Blue White colony selection

AmpR

Ampicillin resistanceIPTG Isopropyl β-D-1-thiogalactopyranoside Xgal5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

pGEM3Z—in vitro transcription 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

HindIII sites

Phage Cloning vectors

GGATTC

Single stranded DNA needed for sequencingIn vitro mutagenesis

• M13 and Lambda phage vectors

Construction of M13 vector

Introducing MCS in M13

Hybrid plasmids-M13 vector• Phagemids………… pEMBL8

1300 bpHelper phage

Construction of Lambda Phage vectors

5-25 kb fragment

37-52 is the range

Deletion 15 kb 18 available

Insertional and replacement vectors• Fragment of lambda is deleted and religated

insert can be inserted in this unique site• Either selection with LacZ” or cI insertional

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

Insertional vectors

Replacement vector

20 kb fragment, size based on Spi phenotype