1. SEMINAR PRESENTATION ON:
CLONING VECTORS USED IN GENETIC
ENGINEERING
BY
GAINA EMMANUEL
PAS/MCB/16/002
SUPERVISED BY:
Dr. AGWARENZE DAWN IFY
DEPARTMENT OF MICROBIOLOGY,
FACULTY OF PURE AND APPLIED
SCIENCE
FEDERAL UNIVERSITY WUKARI.
2. CONTENTS
Introduction
Features of cloning vector
Types of cloning vector
Plasmid
Nomenclature of plasmid and structure of plasmid
Bacteriophage
Cloning features of bacteriophage
Structure of bacteriophage
Cosmid
Structure of cosmid
3. CONTENT CONTINUATION
Bacteria artificial chromosome
Structure of bacteria artificial chromosome
Yeast artificial chromosome
Human artificial chromosome
Structure of human artificial chromosome
Factors that determine the choice of cloning vectors
Conclusion
references
4. INTRODUCTION
A cloning vector is a DNA molecule that carries
foreign DNA into a host cell, replicates inside a
bacterial (or yeast) cell and produces many copies of
itself and the foreign DNA (Nora et al., 2019).
Cloning vector provides the back bone for the DNA
insert to be reproduced and propagated in a suitable
host .The type of cloning vector to be used is
determined by the type of host cell and objective of
the cloning experiment (Bassalo et al.,2016).
5. FEATURES OF CLONING VECTORS
Origin of replication (Ori)
Contain a genetic marker (usually dominant) for
selection
Multiple cloning sites (MCS)
It should be small in size.
It must also be compatible with the host
organism (Nora et al., 2019).
7. PLASMID
Extra-chromosomal, self replicating, double
stranded, closed circular DNA molecules present
in bacterial cell.
Useful for cloning DNA inserts less than 20 kb
(kilo base pairs). Inserts larger than 20 kb are lost
easily in the bacterial cell.
Chang and cohen first proved the use of plasmid
as gene cloning vector (Jahn et al.,2016).
8. NOMENCLATURE AND STRUCTURE OF
PLASMID CLONING VECTOR
NOMENCLATURE
The plasmid pBR322 has the
following elements:
p= plasmid
B= Bolivar (name of the scientist)
R= Rodriguez (name of the
scientist)
322= number of plasmid
discovered in the same lab
STRUCTURE
Figure 1: structure of a plasmid
cloning vector showing its features
Source: (Jahn et al., 2016)
9. BACTERIOPHAGE
Bacteriophages are natural vectors that transduce
bacterial (Escherichia coli)DNA from one cell to
another.
The bacteriophages used for cloning are the
Phage Lambda (λ) And M13 phage
To allow foreign DNA to be inserted into phage,
phage cloning vectors need to have some
nonessential genes deleted .
The Phages are simple in structure and consist of
DNA molecule having several gene for phage
replication which is surrounded by a capsid made
up of proteins.
It consist of an icosahedral head and flexible tail
(Christensen 2001).
10. BACTERIOPHAGE
CONTINUATION.
Lambda phage DNA is a linear DNA duplex
with cohesive single stranded extensions
which are complementary to each other and
consist of 12 nucleotides (cos sites)
Free end of the cos sites has a 5’ phosphate
group (Christensen 2001).
11. CLONING FEAUTURES OF
BACTERIOPHAGE
the genes related in terms of function are
clustered together in the genome and allows
the genes to be switched on and off as a
group rather than individually
The linear double stranded DNA molecule
has a stretch of 12 nucleotides at its either
ends (cos site)
They can base pair to form a circular DNA
molecule which is important for insertion
into the bacterial genome (Bassalo et
al.,2016).
13. COSMID
Cosmid are hybrids between plasmid and phage
lambda ( plasmid + cos site = cosmid)
Formed by joining ends of a linearized plasmid
DNA with cosites
As with the phage lamba vector, the cosmid vector
encodes the cos sequences required for packaging
of DNA into lamba capsid
Cosmid can contain up to 44kbp of DNA
15. BASCTERIALARTIFICIAL
CHROMOSOME (BAC)
A bacterial artificial chromosome (BAC) are
plasmid (circular DNA molecules) constructed with
the OriV of Escherichia coli F’ factor
BAC can carry DNA inserts of greater than 300 kb,
although 100–200 kb (kilo base) is more typical
due to the limitations of preparing such large DNAs
and transferring them into bacteria
16. BAC CONTINUATION.
The BACs, with their inserted DNA, are
then taken up by bacterial cells. As the
bacterial cells grow and divide, they amplify
the BAC DNA
Bacterial artificial chromosome vectors
(BACs) were developed to permit the
cloning and stable maintenance of large
(100–200 kb) pieces of DNA in Escherichia
coli (Shizuya and Kouros-Mehr 2001)
17. STRUCTURE OF BAC
Figure 4: Structure of Bacterial Artificial
Chromosome
Source: (Shizuya and Kouros-Mehr 2001). ).
18. YEAST ARTIFICIAL
CHROMOSOME (YAC)
YAC is a human engineered DNA molecule use to
clone DNA sequences in yeast cells.
YAC are capable o replicating and being selected in
common bacterial host e.g E.coli as well as in the
budding yeast sacccharomyces cerevisiae
YAC is an artificially constructed chromosome that
contains:
Centromere
Telomeres
Autonomous replicating sequences (ARS) (Arnak et
al., 2012)
19. STRUCTURE OF YAC
Figure 5: structure of yeast artificial chromosome
Source: (Arnak et al., 2012).
20. HUMAN ARTIFICIAL
CHROMOSOME (HAC)
A human artificial chromosome (HAC) is a that can act
as a new microchromosome in a population
of human cells of human chromosome
Discovered by H williard in 1997
instead of 46 chromosomes, the cell could have 47 with
the 47th being very small, roughly 6–10 mega
base (Mb) in size instead of 50–250 Mb for natural
chromosomes, and able to carry new genes introduced by
human researchers (Logsdon et al., 2019).
21. STRUTURE OF HAC
Figure 6: structure of human artificial chromosome
Source: (Logsdon et al., 2019).
22. FACTORS THAT DETERMINES
THE CHOICE OF CLONING
VECTOR
Insert size
Vector size
Restriction sites
Copy number
Cloning efficiency
Ability to screen for inserts
23. APPLICATION OF CLONING
VECTORS
Generating whole DNA libraries of the genomes of
higher organisms
used in modeling genetic diseases to study their effect
on transgenic mice
Use in studying neurological diseases such Alzheimer’s
disease
They are useful for the construction of genomic
libraries
In genomic analyses it helps in determining
phylogenetic lineage between species
24. CONCLUSION
There are different types of cloning vectors used in
genetic engineering
These include plasmid, bacteriophage, cosmid,
bacterial artificial chromosome, yeast artificial
chromosome and human artificial chromosome
The best cloning vector is chosen for use according
to the purpose of use and size of the DNA fragment
to be carried.
25. REFFERENCES
Arnak, R., Bruschi, C. V. and Tosato, V. (2012). Yeast Artificial Chromosomes.
In eLS, (Ed.). 3:1-3
Bassalo, M.C., Liu, R., and Gill, R.T. (2016). Directed evolution and synthetic
biology applications to microbial systems. Curr Opin Biotech nol. 39: 126–133.
Christensen A.C, (2001). Bacteriophage lamda-based expression vectors.
Molecular biotechnology. 17(3): 219-224.
John E. Cronan, (2003). Cosmid-Based System for Transient Expression and
Absolute Off-to-On Transcriptional Control of Escherichia coli Genes
Departments of Microbiology and Biochemistry, University of Illinois, Urbana,
Illinois. 185 (2):23-45.
Jahn, M., Vorpahl, C., Hübschmann, T., Harms, H., and Müller, S. (2016) Copy
number variability of expression plasmids determined by cell sorting and droplet
digital PCR. Microbiology Cell Fact. 15: 1–12.
26. REFERENCES CONTINUATION
Kouprina, N., Earnshaw, W. C., Masumoto, H., and Larionov, V. (2013). A new
generation of human artificial chromosomes for functional genomics and gene
therapy. Cellular and molecular life sciences. 70(7): 1135–1148.
Nora, L. C., Westmann, C. A., Martins-Santana, L., Alves, L. F., Monteiro, L.,
Guazzaroni, M. E. and Silva-Rocha, R. (2019). The art of vector engineering:
towards the construction of next-generation genetic tools. Microbial biotechnology.
12(1): 125–147.
Shizuya, H. and Kouros-Mehr, H. (2001). The development and applications of the
bacterial artificial chromosome cloning system. Keio J Med. 50 (1):26-30.
Logsdon, G. A., Gambogi, C. W., Liskovykh, M. A., Barrey, E. J., Larionov, V.,
Miga, K. H., Heun, P. and Black, B. E. (2019). Human Artificial Chromosomes that
Bypass Centromeric DNA. Cell. 178(3): 624–639.
