vectors types prokaryotic and eukaryotic prokaryotic vectors= 1. ti-plasmid 2. pUC18 3. pBR322 4. lambda phage Eukaryotic vectors= 1.BAC 2. YAC by Malaka Madhusudhana Reddy Research Scholar Dept.of :: Zoology Yogivemana University, kadapa. Andhrapradesh, India.

1. MALAKA MADHUSUDHANA REDDY Msc
Research scholar
Dept.of :: Zoology
Yogivemana university, Kadapa

2. What is the difference between cloning and expression vectors?
 Cloning vectors are used to increase the number of copies of the cloned gene or to amplify a foreign gene.
Expression vectors are used to increase the expression of the foreign gene product.
 Cloning is the process of producing genetically identical individuals of an organism either naturally or
artificially.
 In nature, many organisms produce clones through asexual reproduction.
 Cloning in biotechnology refers to the process of creating clones of organisms or copies
of cells or DNA fragments (molecular cloning).
 Beyond biology, the term refers to the production of multiple copies of digital media or software.
Cloning

3. A cloning 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.
The vector therefore contains features that allow for the convenient insertion or removal
of a DNA fragment to or from vector, for example by treating the vector and the foreign
DNA with a restriction enzyme that cuts the DNA.
DNA fragments thus generated contain either blunt ends or overhangs known as sticky
ends, and vector DNA and foreign DNA with compatible ends can then be joined together
by molecular ligation.
After a DNA fragment has been cloned into a cloning vector, it may be
further subcloned into another vector designed for more specific use.

4. 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 clones of recombinant DNA. Such a vector is called Cloning Vector.
Plasmids and phages are the vectors used for cloning purposes, particularly in prokaryotes (bacteria).
Origin of replication (ORI)
This process marks autonomous replication in vector. ORI is a specific sequence of nucleotide in DNA from where
replication starts. When foreign DNA is linked to this sequence then along with vector replication, foreign (desirable)
DNA also starts replicating within host cell.
Selectable Marker
Besides ORI, a cloning vector should have selectable marker gene. This gene permits the selection of host cells which
bear recombinant DNA (called transformants) from those which do not bear rDNA (non-transformants).
Restriction sites
It should have restriction sites, to allow cleavage of specific sequence by specific Restriction Endonuclease. Restriction
sites in E.coli cloning vector pBR322 include HindIII , EcoRI , BamHI , SalI, PvuI, PstI, ClaI etc.

6. Figure 20.3-1
Restriction enzyme
cuts sugar-phosphate
backbones.
Restriction site
DNA
5
5
5
5
5
5
3
3
3
3
3
3
1
Sticky
end
GAATTC
CTTAAG

7. Figure 20.3-2
One possible combination
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
Restriction enzyme
cuts sugar-phosphate
backbones.
Restriction site
DNA
5
5
5
5
5
5
5
5
55
5
5
55
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
1
Sticky
end
GAATTC
CTTAAG
G
G
G
G
AATT CAATT C
C TTAA C TTAA

8. Figure 20.3-3
Recombinant DNA molecule
One possible combination
DNA ligase
seals strands
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
Restriction enzyme
cuts sugar-phosphate
backbones.
Restriction site
DNA
5
5
5
5
5
5
5
5
55
5
5
55
5
5
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3
1
Sticky
end
GAATTC
CTTAAG
G
G
G
G
AATT CAATT C
C TTAA C TTAA

10. cloning vector
Prokaryotic vectors
1. pUC18
2. pBR322
3. Lambda phase
4. Ti-plasmid
Eukaryotic vectors
1. BAC
2. YAC

11. Cloning Vector pUC18

12. pUC18 high copy cloning vector for replication in E. coli, suitable for “blue-white screening” technique.
pUC18 is a small, high copy cloning vector for replication in E. coli.
It has been constructed using the ampicillin resistance gene and the pMB1 origin of replication from pBR322.
It is a circular double stranded DNA and has 2686 base pairs.
The pMB1 of pUC18 differs from the pBR322 origin by a single point mutation and the lack of the rop
gene (also known as repressor of primer), leading to a high copynumber.
A plasmid is a circular dsDNA molecule a few hundred or thousand base pairs in circumference.
The artificial plasmid pUC18 has been genetically engineered to include (1) a gene for antibiotic
resistance to Ampicillin (ampR), and (2) a gene (and its promoter) for the enzyme beta-galactosidase (lacZ).
The lacZ gene contains a (3) polylinker region, with a series of unique restriction sites found nowhere else in
the plasmid.
Digestion with any one of these endonucleases will make a single cut that linearizes the circular
plasmid DNA, and allow it to recombine with foreign DNA that has been cut with the same endonuclease.

13. In the presence of IPTG, transformants expressing both fragments of the ßgalactosidase
will form a functional enzyme and can be detected as blue colonies on agar plates
containing X-Gal.
Cloning into the multiple cloning site will lead to a nonfunctional N-terminal fragment of
the ß-galactosidase and to the abolishment of α-complementation.
White colonies will form on X-Gal/IPTG plates.

14. Cloning Vector pBR322

15. pBR322 is a plasmid and was one of the first widely used E. coli cloning vectors.
Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after
the postdoctoral researchers who constructed it.
The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez."Cloning into the multiple cloning site will lead to a
nonfunctional N-terminal fragment of the ß-galactosidase and to the abolishment of α-complementation
pBR322 is 4361 base pairs in length and has two antibiotic resistance genes – the gene bla encoding
the ampicillin resistance (AmpR) protein, and the gene tetA encoding the tetracycline resistance (TetR) protein.
It contains the origin of replication of pMB1, and the rop gene, which encodes a restrictor of plasmid copy number.
The plasmid has unique restriction sites for more than forty restriction enzymes. Eleven of these forty sites lie within the
TetR gene.
There are two sites for restriction enzymes HindIII and ClaI within the promoter of the TetR gene. There are six
key restriction sites inside the AmpR gene.

16. The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the
TetR gene. The AmpR gene is penicillin beta-lactamase.
Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by
the ligation of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite
strand and initiates transcription in the direction of the tetracycline resistance gene.
A large number of other plasmids based on pBR322 have since been constructed specifically designed for a wide variety
of purposes.
Examples include the pUC series of plasmids.Most expression vectors for extrachromosomal protein expression
and shuttle vectors contain the pBR322 origin of replication, and fragments of pBR322 are very popular in the
construction of intraspecies shuttle or binary vectors and vectors for targeted integration and excision of DNA from
chromosome

18. Lambda phage was first isolated by Esther Lederberg in 1950 from Escherichia coli. It has been an intensely studied
organism, and has been a useful tool in molecular biology.
Lambda phage can be used for cloning of recombinant DNA, the use of its site specific recombinase (int) for the shuffling
of cloned DNAs by the 'Gateway' method, and in recombineering.
Bacteriophage Lambda was studied by Allan Campbell in 1962 who studied the phage's integration process.
Lambda phage is a virus particle consisting of a head made up of double-stranded linear DNA as its genetic material, and
a tail.
It infects the host cell by injecting its own DNA through the tail at which point the phage will enter the lytic or lysogenic
pathway. Large segments of the 48 kilobase pair DNA of the lambda phage are not essential for productive infection and
can be replaced by foreign DNA, thus making lambda phage an ideal vector
He observed that lambda phage had a unique characteristic what some phages would infect and reproduce in some
strains of E. coli while other strains seemed immune.

19. Its linear genome was observed to form a circle via complementary base pairing when entering a host cell. The genome
was able to act in such a peculiar manner due to the presence of single stranded areas called cohesive sites (cos).
These cos sites are able to base pair, and the nature of these sites are the same as those produced by restriction
endonucleases. (Ex: EcoR1 produces "sticky ends").
This circular phage DNA is able to recombine with the host cell DNA via attachment sites. Progeny of the phage with the
addition of some bacterial gene take place by the lysogenic pathway. What makes lambda phage extremely advantageous
is the fact that it can destroy its host (lytic pathway) or it can become part of its host (lysogenic pathway).
Studies on the control of these alternative cycles have been very important for our understanding of the regulation of
gene transcription.

20. Restriction: Phages grown in one bacterial cell fail to grow in other bacterial strains. The
degradation of phage lambda DNA was observed in certain bacterial strains. This was
later found to be a result of restriction endonucleases which would cut the phage DNA
after detecting it as being foreign. Thus, restriction endonucleases was termed because
of its ability to restrict foreign DNA.
Modification: Phages are modified such that they can be grown normally in other
bacterial strains. An example of modification is the methylation of lambda phage DNA so
that it would not be cut by restriction endonuclease.

21. Tumor inducing plasmids (Ti plasmid) are double stranded circular DNA present in
Agrobacterium tumefaciens.
Agrobacterium is a Gram negative soil bacterium which infects dicotyledonous plants.
This plasmid is denatured at higher temperatures and loses tumorgenic properties.
Agrobacterium tumefaciens infects damaged plant tissues, induces the formation of a
plant tumor called crown gall.
The entry of bacterium in to plant tissues is facilitated by the release of certain phenolic
compounds (acetosyringone) by the damaged site
Ti Plasmid

24. Virulence Region
Genes in the virulence region are grouped into the operons virABCDEG, which code for the enzymes
responsible for mediating transduction of T-DNA to plant cells.
virA codes for a receptor which reacts to the presence of phenolic compounds such as acetosyringone,
which leak out of damaged plant tissues.
virB encodes proteins which produce a pore/pilus-like structure
virC binds the overdrive sequence.
virD1 and virD2 produce endonucleases which target the direct repeat borders of the T-DNA segment,
vir E Binds to T-strand protecting it from nuclease attack, and intercalates with lipids to form channels in
the plant membranes through which the T-complex passes, beginning with the right border.
virG (TRANSCRIPTIONAL FACTOR) activates vir-gene expression after binding to a consensus sequence,
once it has been phosphorylated by virA.

25. Application of Ti plasmid
Plant transformation technique using Agrobacterium:
Agrobacterium mediated technique is the most widely used for the transformation of plants and
generation of transgenic plants.
During transformation, several components of the Ti plasmid enable effective transfer of the genes of
interest into the plant cells. These include:
T-DNA border sequences, which demarcate the DNA segment (TDNA) to be transferred into the plant
genome.
vir genes (virulence genes), which are required for transferring the T-DNA region to the plant but are not
themselves transferred, and
modified T-DNA region where the genes that cause crown gall formation are removed and replaced with
the genes of interest.

27. BAC (Bacterial artificial chromosome)

28. RepE: for plasmid replication and regulation of copy number.
ParA and parB: for partitioning F plasmid DNA to daughter cells during division and ensures stable
maintenance of the BAC.
Selectable marker: for antibiotic resistence; some BACs also have lacZ at the cloning site for blue/white
selection.
T7 & Sp6: phage promoters for transcription of inserted genes.
OriS: the origin of replication

31. APPLICATION OF BAC
BACs are being greatly used in modeling genetic diseases in order to study their effects in the
experimentation on transgenic mice.
BAC have been used to study neurological diseases such as Alzheimer’s disease or in the case of down
syndrome.
The genome of several large DNA Viruses and RNA viruses have been cloned as BACs. These constructs are
referred to as “infectious clones”.

32. YEAST ARTIFICIAL CHROMOSOME (YAC)
First described in 1983 by Murray and Szostak.
Yeast artificial chromosomes is a human engineered DNA molecule used to clone DNA sequences in yeast cells.
YACs are shuttle vectors capable of replicating and being selected in common bacterial hosts such as E.coli as well as in
the yeast S. cerevisiae.
They are capable of carrying approximately upto 1000 kbp of inserted DNA sequence.
Many different YAC plasmids exist, such as pYAC3 & pYAC4.

33. COMPONENTS OF YAC
The vector contains several elements of typical yeast chromosomes including:
CEN: A yeast centromere, that ensures chromosome partitioning between two daughter cells and a selective
marker genes.
ARSI: Yeast autonomously replicating sequence
TEL: Yeast telomere
Yeast selectable marker such as URA3 & TRP1 and Bacterial selectable marker
CONSTRUCTION OF YAC
A YAC is built using an initial circular DNA plasmid, which is typically cut into a linear DNA molecule using
restriction enzymes.
DNA ligase is then used to ligate a DNA sequence or gene of interest into the linearized DNA, forming a
single large, circular piece of DNA.

36. APPLICATION OF YAC
Appropriately modified YACs can be utilized in many different organisms, for cloning or
genome analysis.
Chromosomal translocation (chromosome abnormality that occurs due to rearrangement
of parts among non homologous chromosomes) can be studied by means of disposable
YACs that do not contain genetic informationnecessary for cell function.
To identify essential mammalian chromosomal sequences necessary for the future
construction of specialized mammalian artificial chromosomes(MACs).

38. M.MADHUSUDHANA REDDY Msc