Vectors are DNA molecules capable of self-replication that are used to introduce foreign DNA into host cells. Common vectors include plasmids, bacteriophages, cosmids, and artificial chromosomes. Vectors have features like origins of replication, selectable markers, and cloning sites that allow for replication and identification of recombinant DNA. Popular E. coli cloning vectors include the plasmids pBR322, pBR327, and pUC8, as well as the bacteriophages lambda and M13. Hybrid vectors combine characteristics of plasmids and phages, while artificial chromosomes can accommodate larger DNA fragments.

1. VECTOR
VECTOR PREPARATION AND VECTOR CLONING
presented by
Desika.J
Msc agri -1st YEAR

2. VECTORS
 Small DNA molecule capable of self replication.
 Cloning vehicle or Cloning DNA.
 Carrier of DNA fragment.
 E.g. Plasmid, Phage, Hybrid vector, Artificial Chromosomes

3. CHARACTERISTICS
 Self replication, multiple copies
 Replication origin site.
 Cloning site.
 Selectable marker gene.
 Small size.

4.  Low molecular weight.
 Easily isolated & purified.
 Easily isolated into host cell
 Control elements – promoter, operator, ribosome binding site

5. Preparation of vector DNA
 The cloning vector is treated with a restriction endonuclease to
cleave the DNA at the site where foreign DNA will be inserted.
 The restriction enzyme is chosen to generate a configuration at the
cleavage site that is compatible with the ends of the foreign DNA
(see DNA end).
 Typically, this is done by cleaving the vector DNA and foreign DNA
with the same restriction enzyme, for example EcoRI.

6.  Most modern vectors contain a variety of convenient cleavage sites that are
unique within the vector molecule (so that the vector can only be cleaved at a
single site) and are located within a gene (frequently beta-galactosidase)
whose inactivation can be used to distinguish recombinant from non-
recombinant organisms at a later step in the process.
 To improve the ratio of recombinant to non-recombinant organisms, the
cleaved vector may be treated with an enzyme (alkaline phosphatase) that
dephosphorylates the vector ends.
 vector molecules with dephosphorylated ends are unable to replicate, and
replication can only be restored if foreign DNA is integrated into the cleavage
site.

8. Cloning vector

9. VECTOR
 Plasmid
 Bacteriophages
 Cosmid
 Yeast Cloning Vectors
 Ti & Ri Plasmids
TARGET HOST CELL
 Bacteria,Streptomyces
 Bacteria
Bacteria
Yeasts
Transformation of cloned gene
in higher plants.

10. AGENTS USED AS VECTORS
 PLASMIDS
 BACTERIOPHAGES
 COSMID
 ARTIFICIAL CHROMOSOME VECTORS
In 1973, Cohen described first successful construction of recombinant
vector. Plasmid PSC101 - Ecoli

11. PLASMID
 Extra chromosomal DNA molecules.
 Self replicating.
 Double stranded
 Short sequence of DNA.
 Circular DNA molecules
 Found in prokaryotes.

12. CHARACTERISTICS
 Minimum amount of DNA
 Two suitable markers for identification
 Single restriction site.
 More restriction enzyme.
 Relaxed replication control.
 Restriction endonuclease enzyme.

13. Three types of plasmids
Fertility plasmids:-
can perform
conjugation.
Resistance
plasmids:-
contain genes that
build a resistance
against antibiotics
or poisons
Col plasmids:-
contain genes that
code for proteins
that can kill
bacteria.

14. Examples
of
plasmids pB
R3
22
pBR327
pBR325
pBR328
pUC8
pUC9
pUC12
pUC13
pGEM3Z

15. PBR322
 Cloning Vector
 15 copies.
 Reconstructed plasmid.
 Derived from Ecoli plasmid- ColE1.

16. PBR322 – 4362 base pairs
 P – denotes Plasmid
 B – Scientific Boliver
 R - Rodriguez 322 – number given to distinguish.
 Ampicillin resistance gene derived from RSF2124.
 Tetracycline resistance gene from PSC101.
 Origin of replication from PMB1.
 Two selectable markers
Ampr
tetr

17. Structure of E.Coli plasmidcloning vector pBR322

18. PBR327
Derived from PBR322.
 30 – 40 copies.
 Expression plasmid.
Ampilicin resistance gene.
Tetracycline resistance gene
 Deletion of nucleotide between 1427 & 2516

19. pUC8
 Popular Ecoli cloning vector
 Derivative of pBR322.
 Two parts derived
 Ampicillin resistance gene.
 ColEI – origin of replication
 2700 base pairs
 lac Z gene derived from Ecoli.
Polylinker sequence having unique restriction sites lies region in lac

20. PHAGE
 Cloning large DNA fragment
 Linear Phage molecule.
 Efficient than plasmid.
 Used in storage of recombinant DNA.
Commonly used Ecoli phages :- λ phage, M13
Phage

21. BACTERIOPHAGE VECTORS
 Cloning Vectors.
 It infects bacteria.
 Commonly used Ecoli phages :-
λ phage
M13 Phage

22. Lambda phage
 vector Genome size is 48,502 bp.
 High transformation efficiency. 1000 times more efficient than the
plasmid vector.
 Origin of replication
 Genome linear in head. Single- stranded protruding cohesive ends of
12 bases.
 Cos site – site of cleavage of phage DNA

24. Phage M13 Vectors
 Filamentous bacteriophage of Ecoli.
 Used for obtaining single stranded copies
 DNA sequencing. Single stranded.
 Inside host cell become double stranded

26. HYBRIDVECTOR
 Component from both plasmid & phage chromosomes.
 Helper phage provided.
 Developed in 1978 by Barbara Hohn & John Collins
 30 – 40 Kb
 Origin of replication, cloning site, marker gene, DNA cos site.
 Smaller than plasmid.
 Use – construction of genomic libraries of eukaryotes.
 e.g. Cosmid

28. COSMIDS
Combine parts of the lambda chromosome with parts of plasmids. Contain
the cos sites of λ and plasmid origin of replication. Behave both as plasmids
and as phages. Cosmids can carry up to 50 kb of inserted DNA.
Structure of Cosmid
Origin of replication (ori). Restriction sites for cleavage and insertion of foreign DNA.
Selectable marker from plasmid. A cos site - a sequence yield cohesive end (12
bases). Ampicillin resistance gene (amp)

29. ARTIFICIAL CHROMOSOME
 Linear or Circular.
 1 0r 2 copies per cell.

30. Bacterial
Artificial
Chromosome
(BAC)
Yeast Artificial
Chromosome
(YAC)
P1 derived
artificial
chromosome
(PAC)
Mammalian
Artificial
Chromosome
(MAC)
Human
Artificial
Chromosome
(HAC)

31. YAC – Cloning in yeast
BAC & PAC – Bacteria
MAC & HAC – Mammalian & Human cells

32. BACTERIAL ARTIFICIAL CHROMOSOME
 1st BAC Vector – PBAC108L.
 Cloning of large regions of eukaryotic genome.
 Origin of replication from bacterium Ecoli F -factor.
 BAC vectors are pBACe3.6, pBeloBAC11.
 Used in analysis of genomes
 Host for BAC is mutant strain

33. YEASTARTIFICIAL CHROMOSOME
Linear Plasmid Vector
Clone large DNA segment ( 100 – 1400kb).
Occurring two forms:-
Circular – grows in bacteria.
Linear – multiplies in yeast cells.
pYAC3 - first YAC developed.
It contains :-
ARS sequence – replication
CEN4 sequence – centromeric function
TRP1 & URA3 – 2 selectable markers
Use – mapping complex eukaryotic chromosome .