It contain basic of vector construction or commally use vector and their some application....

1. Prepared By :- Umang Vanani
CONSTRUCTION OF VECTOR

2. WHY IS A VECTOR REQUIRED ?
During cloning we transfer a particular gene which
has a desired characteristic which we want. For that, we
require a vector(vehicle) which helps us to transfer the
gene to the bacteria.

3. WHAT IS A VECTOR ?
A vector is a small piece of DNA, taken from a virus, a
plasmid, or the cell of a higher organism, that can be stably
maintained in an organism, and into which a foreign DNA
fragment can be inserted for cloning purposes.

4. TYPES OF VECTOR
1. CLONING VECTOR
2. EXPRESSION VECTOR

5. EXPRESSION VECTOR
The vector design for expression of production protein
specified by the DNA insert is called EXPRESSION
VECTOR.

6. EXPRESSION VECTOR
TWO TYPE :-
1. PROKARYOTIC EXPRESSION VECTOR
Bacterial expression system (e.g. E.coli)
2. EUKARYOTIC EXPRESSION VECTOR
Yeast expression system (e.g. S.cervesiae)
Viral expression system (e.g. Baculovirus)
Mammalian cell expression system

7. CLONING VECTORS
A cloning vector is a DNA molecule in which foreign DNA
can be inserted or integrated and which is further capable
of replicating within host cell to produce multiple clone of
recombinant DNA.

8. CLONING VECTORS
TYPES :-
● PLASMID
● BACTERIOPHAGE
● COSMID
● BACTERIAL ARTIFICIAL CHROMOSOME
● YEAST ARTIFICIAL CHROMOSOME
● HUMAN ARTIFICIAL CHROMOSOME

9. PROKARYOTIC EXPRESSION VECTOR
ADVANTAGES :-
● Gene expression can be easily contolled.
● Easy to grow with high yields.
● Easy to manipulate.
● product can be designed for secretion into the growth
media.

10. PLASMID
● Plasmid vectors can be
used for cloning of DNA
fragments of size generally
ranging from 0.1 to 10 kb.
● The most common
examples of plasmid
cloning vectors are
pUC18/19, pBluescript,
pGEX, pET series etc.

11. PLASMID VECTOR pBR 322
CONTAIN
● Ampicillin resistance
gene.
● Tetracycline resistance
gene.
● origin of replication.
● Eco RI site.

12. BACTERIOPHAGE
● Bacteriophage is a genetically
complex but very extensively
studied virus of E.coli.
● The DNA of phage, is a linear
duplex molecule of 48502 bp
(~49kb) in length.

13. BACTERIOPHAGE
● The DNA isolated from virus particles is a double
stranded linear molecule with short complementary
single stranded projections of 12 nucleotides its 5’ ends.
● These cohesive termini, also referred to as COS site,
allow the be DNA to be circularized after infection of the
host cell.

14. COSMID
● Cosmid vectors (5-7 kb) are
hybrid between plasmids and
phages. They contain antibiotic
genes and replication origin
from plasmids and ‘cos’ sites
from phages which are
necessary for packaging of
DNA.
● These vectors can clone larger
fragments of size ranging from
35 to 50 kb.

15. LAMBDA PHAGE
● Lambda phage can also be
used as vector for cloning
gene of interest of size
ranging from 8-25 kb
fragments.

16. LAMBDA PHAGE
● This is based on lambda phage genome which is 48,502
bp with a central 33% (stuffer region) which is not critical
for lytic growth and can be replaced with 8-25 kb gene
inserts e.g. gt11, charon phages, lambda ZAP vectors.
Lambda vectors can be introduced into cells at a very high
efficiency.

17. BACTERIAL ARTIFICIAL CHROMOSOME
● BAC vectors contain
sequences from the E. coli
F plasmid and they have
the ability to clone up to
75 - 200 kb fragments.

18. YEAST ARTIFICIAL CHROMOSOME
● YAC vectors contain sequences
required to replicate and
maintain chromosome in
budding yeast and contain a
yeast origin of replication, a
centromere, and a telomere at
each end.
● These vectors are able to clone
very large fragments of >2,000
kb inserts (2 Mb).

19. CONSTRUCTION OF VECTOR
● The first requirement for construction of vector is an
extrachromosomal self replicating circular plasmid.
● A restriction enzyme is required to cut the restriction site
where the desired gene is attached.
● A DNA Ligase is required to join the gene in plasmid at
restriction site.

21. VECTOR
Four common properties of vector
1. Ability to promote autonomous replication.
2. Contain a genetic marker(usually dominant) for
selection.
3. Unique restriction sites to facilitate cloning to insert
DNA.
4. Minimum amount of non-essential DNA to optimize
cloning.

22. ORIGIN OF REPLICATION
● Origin of replication is a DNA
segment recognized by the
cellular DNA replication
enzyme.
● Without replication origin,
DNA can not be replicated in
the cell.

23. SELECTIVE MARKER
● Selective marker is required for
maintenance of plasmid in the
cell.
● Because of the presence of the
selective marker, the plasmid
becomes useful for the cell.

24. SELECTIVE MARKER
● Under selective conditions, only cells that contain
plasmid with selectable marker can survive.
● Gene that confers resistance to various antibiotic are
used.
● Genes that make cells resistance to ampicillin,
neomycin or chloramphenicol are used.

25. RESTRICTION ENZYME
Mostly Endonuclease is used for cutting of particular
selected gene from the DNA and cut the same sequence in
the plasmid for the attachment.
Two types of cut is done by the enzyme “blunt cut” and
“sticky cut”.
sticky cut is very specific and unique.

26. INJECTION INTO CELL
● Applying small heat shock or small electric shock to the
cell creates small pores in the cell membrane.
● Through these small pores, vector is injected in the cell.

28. CHOICE OF EXPRESSION VECTORS
● Strong promoter
● Intact ORF
● Ribosomal binding site
● Termination sequence

29. CHOICE OF CLONING VECTORS
● A vector should be small and easily prepared.
● Capable of autonomous replication in the host cell.
● It should have one target site only for any restriction
enzyme(that has been used in to make DNA fragments.
● The vector should show Antibiotic resistance.
● The vector must have a marker permitting recognition.

30. Maximum DNA insert accommodate by
different cloning vectors…
Types Size of cloned DNA(kb)
Plasmid 20
Lambda Phage 25
Cosmid 45
BAC 300
YAC 1000

31. APPLICATION OF VECTOR
•The construction of expression libraries.
•The analysis of gene function at protein level.
•The commercial production of proteins.
•The production of antibodies.
•For in vivo studies of the protein.
EXPRESSION VECTOR

32. APPLICATION OF VECTOR
•To amplify the clones of desired DNA segment
•Allow in vitro production of RNA copies of desired DNA
insert.
•Generates single stranded copies of DNA insert
•Construction of genomic DNA libraries and cDNA libraries.
CLONING VECTOR