vectors in rDNA technology

1. VECTORS
SHIVA'S

2. PHAGES
• Derivatives of phage have been developed as cloning vectors since the early days of
gene technology.
• The phage derivatives are considered to be the most suitable cloning vehicles for
cloning genomic eukaryotic DNA because of the following advantages over the
plasmids.
 Thousands of phage plaques can be obtained in a single petri dish.
 Selection by DNA-DNA hybridization is possible.
 In vitro packaging into empty phage head is possible thus increasing phage
infectivity
Size selection of the packaged DNA is possible.
 Millions of independently cloned virus particle can be constituted to form a gene
library.
SHIVA'S

3. BACTERIOPHAGES • 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.
• The DNA isolated from virus particles is a double
stranded linear molecule with short
complementary single stranded projections of 12
nucleotides at its 5’ ends.
• These cohesive termini, also referred to as cos
sites, allow the DNA to be circularized after
infection of the host cell.
SHIVA'S

4. GENERAL STRUCTURE OF BACTERIOPHAGE
• The genetic map of phage comprises approximately 40 genes which are organized in
functional clusters.
• Genes coding for head and tail are proteins (genes A-J) are on the left of the linear
map.
• The central region contains genes, such as int, xis, exo etc., which are responsible
for lysogenisation i.e the process leading to the integration of viral DNA and other
recombination events.
• Much of this central region is not essential for lytic growth.
• Genes to the right of the central region comprise six regulatory genes, two genes (O
and P) which are essential for DNA replication during lytic growth and two more
genes (S and R) which are required for the lysis of the cellular membranes.
SHIVA'S

5. • In the phage DNA, larger central region is not essential for phage growth and
replication.
• This region of phage can be deleted or replaced without seriously impairing
the phage growth cycle.
• Using this non-essential region of phage, several phage vector derivatives have
been constructed for efficient gene cloning.
SHIVA'S

6. TYPES OF PHAGE VECTORS
• Wild type phage DNA itself cannot be used as a vector since it contains too many
restriction sites.
• Further, these sites are often located within the essential regions for phage's growth
and development.
• From these wild phages, derivatives with single target sites and two target sites
have been synthesized.
• Phage vectors which contain single site for the insertion of foreign DNA have been
designated as Insertional vectors;
• vectors with two cleavage sites, which allow foreign DNA to be substituted for the
DNA sequences between those sites, are known as replacement vectors.
SHIVA'S

7. INSERTIONAL VECTORS
• A large segment of the non-essential region has been
deleted, and the two arms ligated together.
• An insertion vector possesses at least one unique
restriction site into which new DNA can be inserted.
• Two popular insertion vectors are:
• Egt10 : which can carry up to 8 kb of new DNA,
inserted into a unique EcoRI site located in the cI
gene.
• Insertional inactivation of this gene means that
recombinants are distinguished as clear rather than
turbid plaques.
• EZAPII : insertion of up to 10 kb DNA into any of 6
restriction sites within a polylinker inactivates the
lacZ′ gene carried by the vector.
• Recombinants give clear rather than blue plaques on
X-gal agar.
SHIVA'S

8. E.COLI/ REPLACEMENT VECTORS
Examples: EMBL3 and  DASH.
A representative scheme for cloning:
1. The  vector DNA is cleaved with
BamH1 and the long (19 kb) and short
(9 kb) arms are purified;
2. The target fragments are prepared by
digestion, also with BamH1 or a
compatible enzyme (Sau3A);
3. The target fragments are treated with
alkaline phosphatase to prevent them
ligating to each other;
4. The  arms and the target fragments
are ligated together at relatively high
concentration to form long linear
products.
B B
 20kb
B
Can not
Parking
infect
E.coli
Long arm
Short
Replace. arm
48.5 kb
SHIVA'S

9. PACKAGING AND INFECTION
The Recombinants that can not be packaged:
1. Ligated  ends which do not contain an insert;
2. The insert is much smaller or larger than the 20 kb;
3. The recombinants with two left or right arms.
in vivo
B
Replication concata-mers
cleave individual  genomes
in vitro
A mixture of phage coat proteins and
the phage DNA-processing enzymes
Packaging:
Packaging
phage
particles
Infection of E. coli
109 recombinants per
mg of vector DNA.
SHIVA'S

10. FORMATION OF PLAQUES
Plaques are the analogs of single bacterial colonies.
Formation:
The infected E.coli cells from a packaging reaction are spread on
an agar plate,
The plate has been pre-spread with uninfected cells, which will
grow to form a continuous lawn.
After incubation, phage-infected cells result in clear areas, that
are plaques, where cycles of lysis and re-infection have
prevented the cells from growing.
E.coli lawn
Plaques
Recombinant  DNA may be purified:
• from phage particles isolated from plaques or
• from the supernatant of a culture infected
with a specific recombinant plaque.
SHIVA'S

11. BACTERIOPHAGE M13
Genome features: Size is small (6.7 kb); Single-stranded;
Circular genome; DNA; Positive-sense.
Infection: M13 particles attach specifically to E.coli sex pili (encoded by a
plasmid called F factor), through a minor coat protein (g3p). Binding of g3p
induces a structural change in the major capsid protein. This causes the whole
particle to shorten, injecting the viral DNA into the host cell.
RF
g6p g3p
g7p
g8p
g9p
Host
enzymes
end
ini
SHIVA'S

12. E.COLI/M13 PHAGE
VECTORS
Structure:  The phage particles
contain a 6.7 kb circular ssDNA. 
After infection of a sensitive E. coli
host, the complementary strand is
synthesized, like a plasmid, and the
DNA replicated as a dsDNA, the
replicative form (RF).
Features:  The host cells can continue
to grow slowly.
• ssDNA: The single-stranded forms
are continuously packaged and
released from the cells as new phage
particles. ssDNA has a number of
applications, including  DNA
sequencing and  site-directed
mutagenesis.
• dsDNA: The RF (dsDNA) can be
purified in vitro and manipulated
exactly like a plasmid.
SHIVA'S

13. CLONING IN M13
Purpose: When the single-stranded DNA of a fragment is required, a M 13
vector can be used as a common cloning tool.
Preparation of ssDNA:
1. Cloning: standard plasmid cloning method can be used to incorporate
recombinant DNA into M13 vectors;
2. Transformation: the M13 then infects sensitive E. coli cells;
3. Plating: the host cells grow to form the plaques;
4. Isolation: the ssDNA may then be isolated from phage particles in the
growth medium of the plate.
Screening: Blue-white screening using MCSs and lacZ' has been engineered
into M13 vectors.
Examples: The M13mpl8 and M13mp19, which are a pair of vectors in which
the MCS are in opposite orientations relative to the M13 origin of
replication.
SHIVA'S

14. HYBRID PLASMID-M13 VECTORS
Definition: A number of small plasmid vectors, for example pBlue-script, have
been developed to incorporate M13 functionality.
Structure: They contain both plasmid and M13 origins of replication, but do not
possess the genes required for the full phage life cycle.
Working ways:
1. Plasmid way: they normally propagate as true plasmids, and have the
advantages of rapid growth and easy manipulation of plasmid vectors;
2. Phage way: they can be induced to produce single-stranded phage particles
by co-infection with a fully functional helper phage, which provides the gene
products required for single-strand production and packaging.
SHIVA'S

15. COSMIDS
• Cosmids are hybrids between a phage DNA molecule and a
bacterial plasmid, and their design centers on the fact that the
enzymes that package the  DNA molecule into the phage
protein coat need only the cos sites in order to function.
• The in vitro packaging reaction works not only with  genomes,
but also with any molecule that carries cos sites separated by
37–52 kb of DNA.
• A cosmid is basically a plasmid that carries a cos site .
• It also needs a selectable marker, such as the ampicillin
resistance gene, and a plasmid origin of replication, as cosmids
lack all the  genes and so do not produce plaques.
• Instead colonies are formed on selective media, just as with a
plasmid vector.
SHIVA'S

16. • The following table provides a list cosmid vectors
and their structural features.
• Cosmid Size(kb) Cleavage sites Size of
insertion (kb)
• MUA3 4.76 EcoRI/PstI/PvuII/PvuI
40 – 48
• pJB8 5.40 BamHI 32 – 45
• Homer I 5.40 EcoRI/ClaI 30 – 47
• Homer II 6.38 SstI 32 – 44
• pJC79 6.40 EcoRI/ClaI/BamH I
32 – 44
SHIVA'S

17. BACTERIAL ARTIFICIAL
CHROMOSOMES (BAC)
BACs are based on bacterial mini-F
plasmids, which are small pieces of episomal
bacterial DNA that give the bacteria the
ability to initiate conjugation with adjacent
bacteria. They have a cloning limit of 75-300
kb.
SHIVA'S

18. YEAST ARTIFICIAL CHROMOSOMES (YAC)
 YACs are artificial chromosomes that replicate in yeast cells. They consist of Telomeres,
which are ends of chromosomes involved in the replication and stability of linear DNA.
 Origin of replication sequences necessary for the replication in yeast cells.
 A yeast centromere, which is a specialized chromosomal region where spindle fibers
attach during mitosis.
 A selectable marker for identification in yeast cells.
Ampicillin resistance gene for selective amplification.
 Recognition sites for restriction enzymes.
SHIVA'S

19. THE PROCEDURE FOR
MAKING YAC VECTORS IS AS
FOLLOWS
1. The target DNA is partially digested by a
restriction endonuclease, and the YAC vector is
cleaved by restriction enzymes.
2. The cleaved vector segments are ligated with a
digested DNA fragment to form an artificial
chromosome.
3. Yeast cells are transformed to make a large number
of copies.
They are the largest of the cloning vectors, with a
cloning limit of 100-1000 kb, however they have very
low efficiency.
SHIVA'S

20. YEAST/YAC VECTORS
CEN4 is the centromere of chromosome 4
of Yeast. The centromere will segregate
the daughter chromosomes.
ARS is autonomously replicating
sequence, its function is as a yeast
origin of replication.
TRP1 and URA3 are yeast selectable
markers, one for each end, to ensure
the right reconstituted YACs survive in
the yeast cells.
TEL is the telomeric DNA sequence,
which is extended by the telomerase
enzyme inside the yeast cell.
SUP4 is a gene, which is insertionally
inactivated, for a red-white color test,
like blue-white screening in E. coli.
pYAC3
B B
S
SnaBI
BamHI
Function: YAC vectors can accept genomic DNA fragments of more
than 1 Mb, and hence can be used to clone entire human genes.
SHIVA'S

21. SHIVA'S

22. SHUTTLE VECTORS
Definition: They are the vectors that can
shuttle between more than one host,
for example, one is E. coli and the
other is yeast.
Structure and function: Most of the
vectors for use in eukaryotic cells are
constructed as shuttle vectors.
• In E. coli:
• This means that they can survive
and have the genes (ori and ampr )
required for replication and
selection in E. coli.
• In the desired eukaryotic cells:
• They can also survive in the
desired host cells, and let the
target insert sequences take effects.
E.coli
Yeast
SHIVA'S

23. YEAST EPISOMAL PLASMIDS
Structure of YEps
a ori: for replication in E.coli
a ampr: for selection in E.
coli
a 2 origin: for replication in
yest
LEU2: is homologous gene
and a selectable marker in
yeast, involved in leucine
synthesis.
X gene: a shuttle sequence.
ori
ampr
2 origin
X gene LEU2
Function of YEps
• It replicates as plasmids
• It integrates into a yeast
chromosome by homologous
recombination.
YEps
SHIVA'S

24. EXPRESSION VECTORS
• In DNA cloning experiments all the genes cloned are not expressed fully because of weak
promoters in vector DNA.
• This can be dramatically improved by placing such genes downstream of strong promoters.
• An additional problem in maximizing expression of cloned genes in E. coli which is
frequently encountered with genes from a heterologous source is that the gene carries no
translation start signal which can be efficiently recognized by the E. coli translation
system.
• This problem may arise for heterologous genes cloned into any host. Thus, even though the
gene can be transcribed from a promoter within the vector, the resulting mRNA is poorly
translated and little or no protein product will be synthesized.
• In such cases alternative strategies available are fusing the gene to amino terminal region
of vector gene that is efficiently translated in the host or coupling the gene to a DNA
fragment carrying both strong promoter and a ribosomal binding site.
• Vectors with this additional feature are called expression vectors.
SHIVA'S

25. SHIVAS
T7
expressional
vector
E.COLI/T7 EXPRESSION
VECTORS
• Definition of expression vectors:
Cloned geneexpression vector
hostfusion protein.
• Structure
T7
• T7 promoter: a strong promoter;
• RBS: ribosome binding site;
• ATG: translation initiation
condon
• MCS: Multiple cloning sites
• TT: transcription terminator.
• ampr,. ori,
• His-tag: Some expression vectors are
designed to have six histidine codons
that encode a hexahistidine tag at
the N terminus of the expressed
protein, which allows one-step
purification on an affinity column
containing Ni2+.
RBS MCS
TT
ATG

26. INSECT CELL/BACULOVIRUS
Definition: Baculovirus is an  insect virus which
can be used for the overexpression of  animal
proteins  in insect cell culture.
Mechanism:
• Viral promoter: This viral gene has an extremely
active promoter.
• Insect cell culture: The same promoter can be
used to drive the over-expression of a foreign
gene engineered into the baculovirus genome.
Function: This method is being used increasingly
for large-scale culture of proteins of animal
origin, since the insect cells can produce many of
the post-translational modifications of animal
proteins, which a bacterial expression system
cannot.
Baculovirus-infected SF21 cells
SHIVA'S

27. SHIVAS
MAMMALIAN CELL/VIRAL VECTORS
• SV40: This virus can infect a
number of mammalian species.
The SV40 genome is only 5.2 kb
in size.
• Since it has packaging
constraints similar to phage , so
it can be not used for
transferring large fragments.

28. SHIVAS
MAMMALIAN CELL/VIRAL
VECTORS
• Retroviruses: They have a ssRNA genome,
which is copied into dsDNA after infection.
The DNA is then stably integrated into
the host genome by a transposition
mechanism.
• They have some strong promoters, and
they have been considered as vectors for
gene therapy, since the foreign DNA will
be incorporated into the host genome in a
stable manner.