1. Vectors
Dr Muhammad Imran

2. What are plasmids Resistance conferring…..R 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,

6. Pedgree of pBR322

7. Earlier derivative of pBR322
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 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

11. 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

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

13. Construction of M13 vector

14. Introducing MCS in M13

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

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

18. Deletion 15 kb 18 available

20. 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

21. Insertional vectors

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