This document discusses different types of cloning vectors. It describes that vectors are used to carry foreign DNA into host cells. There are two main types of transformation vectors - cloning vectors which are used to increase copies of cloned DNA fragments, and expression vectors which are used to express foreign genes as proteins. Some examples of commonly used cloning vectors include pBR322 and pUC18/19, while examples of expression vectors include pET28 and pRSET vectors. The document then discusses various properties desirable in vectors such as an origin of replication, antibiotic resistance genes, and regulatory elements. It describes different types of vectors including plasmid vectors, bacteriophage vectors, cosmids, BACs/YACs, and mini chromosomes.

1. CLONING VECTORS
Dr. M. THIPPESWAMY

2. Recombinant DNA Technology

3. Vector is an autonomously replicating (inside a host cell)
DNA molecule designed from a plasmid or phage DNA to
carry a foreign DNA inside the host cell.
Transformation vectors are of two types:
• Cloning vector is used increasing the number of copies of a
cloned DNA fragment. Eg., pBR322, pUC18/19
• Expression vector is used for expression of foreign gene
into a protein. Eg., pET28, pRSET-A, B & C
• If a vector is designed to perform equally in two different
hosts, it is called a shuttle vector. Eg., pYES2, Ti-Plasmid
VECTORS

4. Properties of an ideal vector:
A good vector should have the following characteristics:
• Autonomously replicating i.e. should have ori (origin of replication) region.
• Contain at least one selectable marker e. g. gene for antibiotic resistance.
• May contain a scorable marker (β-galactosidase, green fluorescent protein etc.)
• Presence of unique restriction enzyme site.
• Have multiple cloning sites.
• Preferably small in size and easy to handle.
• Relaxed control of replication to obtain multiple copies.
• Presence of appropriate regulatory elements for expression of foreign gene.
• High copy number
The selection of a suitable vector system depends mainly on the size limit of
insert DNA and the type of host intended for cloning or expression of foreign
DNA.

5. Types of vectors
• Plasmid vectors
• Bacteriophage vectors
• Cosmids
• BACs & YACs
• Mini chromosomes

6. • These Plasmids are extrachromosomal circular DNA molecule
that autonomously replicates inside the bacterial cell.
• Cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb)
.
• In their simplest form, plasmids contains a bacterial origin of
replication, an antibiotic resistance gene, and at least one unique
restriction enzyme recognition site which helps in cloning.
PLASMIDS

13. Based on the origin or source of plasmids, they have been divided
into two major classes: such as natural and artificial.
i) Natural plasmids: They occur naturally in prokaryotes or
eukaryotes. Example: ColE1.
ii) Artificial plasmids: They are constructed in-vitro by re-
combining selected segments of two or more other plasmids
(natural or artificial). Example: pBR322.
PLASMIDS

14. Characteristics of natural plasmids

15. Artificial plasmids vectors are classified into two broad types
based on their use:
1. Cloning vector
2. Expression vector
Artificial plasmids
A cloning vector is defined as a vector used for replication of a
cloned DNA fragment in a host cell.
These vectors are frequently engineered to contain “ori” – origin
of replication sites particular to the host organism.
Examples of commonly used cloning vectors are: pUC18,
pUC19, pBluescript vectors etc.

16. Basic components of a plasmid

17. Antibiotic Ampicillin
Mode of
action
Inhibits the cell wall synthesis by dirupting
peptidoglycon cross linking
Resistance
gene
B-galactamase (amp) gene product is secreted
and hydrolyse the ampicillin
Application Amp gene is included on plasmid vector as
positive selection markers
Antibiotic Tetracycline
Mode of
action
Inhibits the binding of aminoacyl tRNA to the
30S ribosomal subunit
Resistance
gene
tet gene product is membrane bound and
prevents tetracycline accumulation by an efflux
mechanism
Application tet gene product is a positive selection marker
on some plasmids
Antibiotics and antibiotic resistance genes used in gene cloning

18. Antibiotic Kenamycin
Mode of
action
Inactivates translation by interfering with
ribosome function
Resistance
gene
Neomycin or amino glycoside phospho
transferase (neo) gene product anactivates
kanamycin by phosphorylation
Application Neo gene is a positive selection marker used in
plasmids
Antibiotic Bleomycin
Mode of
action
Inhibits DNA and RNA synthesis by binding to
DNA
Resistance
gene
The bla gene product binds to bleomycin and
prevents it from binding to DNA
Application Bla gene is a positive selection marker used in
plasmids

19. Antibiotic Hygromycin B
Mode of action Inhibits the translation in prokaryotes and eularyotes
by interferring with ribosome translation
Resistance gene Hygromycin-B-phosphotransferase (hyg) gene
product inactivates hygromycin B by phosphorylation
Application hyg gene is used as a positive selection marker in
eukaryotic cells that are sensitive to hygromycin B
Antibiotic Chloromphenicol
Mode of action Binds to the 50S ribosomal subunit and inhibits
translation
Resistance gene Chloramphenicol acetyl transferase (CM) gene
product metabolises chloramphenicol in the presence
of acetyl CoA
Application CM gene is used as a selectable marker, and as
transcriptional reporter gene of promoter activity in
eukaryotic cells

20. Advantages of Plasmids in Molecular Biology
• Easy to work with - Plasmids are a convenient size
(generally 1,000-20,000 base pairs).
• Self-replicating - Endless number of copies of the plasmid
was obtained by growing the plasmid in bacteria.
• Stable - Plasmids are stable long-term either as purified
DNA or within bacteria (as glycerol stocks).
• Functional in many species and can useful for a diverse set
of applications - Plasmids can drive gene expression in a
wide variety of organisms, including plants, worms, mice
and even cultured human cells.

21. Major Limitation of Cloning in Plasmids
Upper limit for clone DNA size is 12 kb
Requires the preparation of “competent” host cells
Inefficient for generating genomic libraries as overlapping
regions needed to place in proper sequence
Preference for smaller clones to be transformed
If it is an expression vector there are often limitations
regarding eukaryotic protein expression.

22. Examples of Cloning Vector:

23. pBR322
• pBR322 is a widely-used E. coli cloning
vector. It was the first created plasmid in
1977. The p stands for "plasmid" and BR
for "Bolivar" and "Rodriguez",
researchers who constructed it.
• pBR322 is 4361 base pairs in length.
“rep” replicon from plasmid pMB1 which is responsible for replication
of the plasmid.
“rop” gene encoding Rop protein. Rop proteins are associated with
stability of RNAI-RNAII complex and also decrease copy number.
“tet” gene encoding tetracycline resistance.
“bla” gene encoding β lactamase which provide ampicillin resistance.

24. pUC plasmids:
• pUC plasmids are small, high copy
number plasmids of size 2686bp.
• This series of cloning vectors were
developed by Messing and co-
workers in the University of
California. The p in its name stands
for plasmid and UC represents the
University of California.
pUC vectors consists of following elements:
pMB1 “rep” replicon region derived from plasmid pBR322 with single point
mutation (to increase copy number).
“bla” gene encoding β lactamase which provide ampicillin resistance which is
derived from pBR322. This site is different from pBR322 by two point mutations.
E.coli lac operon system.
• “rop” gene is removed from this vector which leads to an increase in copy number.

25. A vector used for expression of a cloned DNA fragment in a host cell is
called as an expression vector.
These vectors are frequently engineered to contain regulatory sequences that
act as promoter and/or enhancer regions and lead to efficient transcription.
Commonly used expression vector series are: pET vectors, pBAD vectors,
pRSET-A, B & C vectors etc.
Expression Vector
For an expression vector following features are essential:
• Promoter: Promoter sequence recognizes the RNA polymerase which is required
for initiation of transcription of gene of interest.
• Terminator: It is a DNA element present at the end of a gene where transcription of
gene ends.
• Ribosome binding site: It is a short nucleotide sequence recognized by the
ribosome as the point at which it should attach to the messenger molecule.
The initiation codon of the gene is always a few nucleotides downstream of this.

26. pET28 vector:
• pET vector system is a cloning and
expression vector system for recombinant
protein production in E.coli. This product is
registered under trademark of Novagen Inc.
• The original pET vector system was
constructed by Studier and colleagues. That
plasmid is developed at Novagen with
enhanced characteristics.
• Target genes are cloned under strong T7 bacteriophage promoter.
• The expression of the target protein is inducible by providing T7 RNA
polymerase in the host cell as an inducing signal.
• Ampicillin and kannamycin resistance genes are available in pET vectors as
selection marker.
• pET28 and pET32 are the most commonly used pET vectors.

27. The pRSET vectors are pUC-
derived expression vectors
designed for high-level
protein expression and
purification from cloned
genes in E. coli.
High levels of expression of
DNA sequences cloned into
the pRSET vectors are made
possible by the presence of
the T7 promoter.
T7 RNA polymerase specifically
recognizes this promoter.
For expression of the gene of interest, it is necessary to deliver T7 RNA polymerase to
the cells by either inducing expression of the polymerase using the gratuitous inducer
isopropyl β-D- thiogalactoside (IPTG), or infecting the cell with phage expressing the
polymerase.
pRSET A B C – Expression vector

28. Vector for cloning Saccharomyces
genes.
The presence of a yeast origin of
DNA replication (ARS) and a yeast
centromere (CEN) allows, stable
replication and segregation in
yeast. Also included is a yeast
selectable marker such as URA3,
which allows a ura3 mutant to
grow on medium lacking uracil.
Typical protocol for constructing a
yeast genomic library.
Components of a typical plasmid shuttle

29. Phage Vectors:
To insert DNA fragments of more than 15 kb, normally plasmids are not
the suitable vehicles, as large inserts may trigger plasmid rearrangement or
affect plasmid replication.
This leads to development of a new class of vectors based on
bacteriophages.
Amongst various
bacteriophages
available such as
λ, T4, T5, and T7
phages; the λ
phage gained
favourable
attention due to its
unique life cycle.

30. λ phage vector
Bacteriophage λ contains ~49kb of DNA and has a very efficient
mechanism for delivering its genome into a bacterium. Two key features
contribute to its utility as a vector to clone larger DNA fragments:
One-third λ genome is nonessential and could be replaced with foreign
DNA. Approximately 24.6kb of λ genome can be deleted, hence
maximum insert size could be upto 26 kb.
Applications:
• DNA sequencing
• Mutagenesis study
• probe generation
• Phage display

31. • Cosmids are medium sized cloning
vectors.
• The cloning capacity of these vectors is
35-45 kbp.
• Cosmid vector are developed by
combining the features of plasmid vector
and bacteriophage vector.
• The first cosmid vector was described by
Collins in 1978.
COSMID VECTOR
In Brief
• A cosmid is a plasmid that contain phage sequence that allows the vector
to be packaged and transmitted to bacteria like phage vector.
Or
• A cosmid is a type of hybrid plasmid that contains a Lambda phage cos
sequence.
Cosmids (cos sites + plasmid = cosmids) DNA sequences are originally
from the lambda phage.

36. THANK YOU