Genetic Engineering: Cloning Vectors

Genetic Engineering
Chapter 3- Cloning Vectors
Hikmet Geçkil, Professor
Department of Molecular Biology and Genetics
Inonu University

Cloning vectors
• Vectors:
– are autonomously replicating DNA molecules that can be used to carry
foreign DNA fragments
– must possess the ability to self-replicate
– must have a selectable characteristic so that transformed cells may be
recognized fromuntransformed cells
– must contain restriction enzyme recognition sites so that DNA
fragments can be cloned into
• Most cloning experiments utilize the bacterium Escherichia coli as
the host for the propagation of cloned DNA fragments, given its:
– ease and cost to grow
– rapid growth (e.g., doubling time 20–30 min)
– well understood genetics
– being safe and having desired mutants
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 2

• Widely distributed
throughout prokaryotes
• Mostly in sizes 1 to 20
kbp.
• Relaxed plasmids at
multiple copies (10–
200), stringent plasmids
at low copies (1–2) per
cell.
• Thus, stringent plasmids
are biger and have more
genes on them.
Geckil
Cloning vectors: plasmids

• The genes carried in plasmids provide bacteria with
genetic advantages, such as antibiotic resistance.
• Most plasmids in common use are based upon the
replication origin of the naturally occurring E. coli
plasmid ColE1, or its very close relative pMB1
• ColE1 is a 6646 bp closed-circular DNA molecule that
encodes a bacteriocin, colicin E1, and a bacteriocin
resistance gene
• Colicin E1 is a transmembrane protein that causes
lethal membrane depolarization in bacteria
Geckil

Types of Plasmids
• Cloning Plasmids - Cloning vectors are simple, often
contain only a bacterial resistance gene, origin and
MCS.
• Expression Plasmids - Used for gene expression. They
contain promoter terminator sequences and the
inserted gene. An expression vector can also include an
enhancer sequence which increases the amount of
protein or RNA produced.
• Gene knock-down Plasmids - Used for reducing the
expression of an endogenous gene. This is frequently
accomplished through expression of an shRNA
targeting the mRNA of the gene of interest.
Geckil

• Reporter Plasmids - Used for studying the function of
genetic elements. They contain a reporter gene (e.g.,,
luciferase or GFP) that offers a read-out of the activity
of the genetic element.
• Viral Plasmids - used in delivery of genetic material
into target cells just like a virus . One can use these
plasmids to create viral particles, such as lentiviral,
retroviral, and adenoviral particles.
Geckil

pBR322, one of the first constructed plasmids
• pBR322 is 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,
"Bolivar" and "Rodriguez".
• It has all the essential requirements
for a cloning vector (i.e., relatively
small size, useful restriction enzyme
sites, an origin of replication, and
antibiotic resistance genes).
Genetic Engineering/Hikmet Geckil Chapter 3: Cloning Vectors 7
Map and important features of pBR322

pUC series of plasmids
• pUC (origin of name:
University of California)
cloning vectors are high copy
number and contain a
multiple cloning site at the
lacZ' region.
• Created by Joachim
Messing and co-workers in
1982.
• Their recombinants can
be verified via blue/white
colony screening using agar
plates containing IPTG and X-
Gal.
• Expression of target DNA is
enabled by the presence of
a lac promoter in the cloning
vectors.
Geckil
Map and important features of pUC18

• The plasmid pBR322 has an origin of replication, or ori, a
sequence where replication is initiated by cellular enzymes.
• The plasmid contains genes that confer resistance to the
antibiotics tetracycline (TetR) and ampicillin (AmpR),
allowing the selection of cells that contain the plasmid
• Several unique recognition sequences in pBR322 are
targets for restriction endonucleases (PstI, EcoRI, BamHI,
SalI, and PvuII), providing sites where the plasmid can be
cut to insert foreign DNA.
• The small size (4,361 bp), generated simply by trimming
away many DNA segments, facilitates its entry into cells and
the biochemical manipulation of the DNA.
Geckil

Genetic Engineering/Hikmet Geckil Chapter 3: Cloning Vectors 10
Use of pBR322 to clone foreign DNA. The
entire procedure is illustrated, including
both positive and negative selection.

Replication origin
• The replication origin
(ORI) is a specific DNA
sequence of 50 – 100
base pairs that must be
present in a plasmid for
it to replicate.
• Host-cell enzymes bind
to ORI, initiating
replication of the
circular plasmid.
Geckil
The parental strands are shown in blue, and
newly synthesized daughter strands are shown
in red. Once DNA replication is initiated at the
origin (ORI), it continues in both directions
around the circular molecule until the
advancing growing forks merge and two
daughter molecules are produced.

Geckil

Vectors based on bacteriophage λ
• The limitation of plasmid vector is the size of DNA that can
be introduced into the cell by transformation.
• This presents problems when you are trying to create a
genomic library of a large genome such as with plants.
• A genomic library contains all of the DNA found in the cell
of the plant (or any organism).
• Thus, you need to use a vector that can accept large
fragments of DNA.
• Examples of these are bacteriophage and cosmid vectors
and more recently yeast artificial chromosomes.
Geckil

Geckil

The life cycle of lambda
• Adsorption - the phage particle binds at a
maltose receptor site of the bacterial cell;
growing the cell in the presence of the sugar
increase the number of receptor sites
• Penetration - DNA is injected into the cell; at this
point it can enter one of two pathways;
– Lysogenic pathway - the phage DNA becomes
integrated into the genome and is replicated along
with the bacterial DNA; it remains integrated until it
enters the lytic pathway
– Lytic pathway - large scale production of
bacteriophage particles that eventually leads to the
lysis of the cell; base pairing at the cos site leads to a
circular molecule
Geckil

• Packaging of the DNA into the lambda phage
head does not require a complete length of
wild type lambda.
• It has been determined that a lambda
molecule that is between 78% and 105% of
wild type length can be packaged. This is from
37 to 53 kb in length.
Geckil

Vectors based on bacteriophage M13
Geckil
Genetic map and important features of bacteriophage vector M13

Cosmids
• Cosmids are plasmid vectors that contain cos
sites.
• The cos site is required for DNA to be
packaged into a phage particle.
• Since phage particles can accept between 38
and 53 kb of DNA and since most cosmids are
about 5 kb, between 33 and 48 kb of DNA can
cloned in these vectors.
Geckil

Hybrid plasmid/phage vectors
• Phagemids, similarly, are not as common as plasmids.
• The most known is pBluescript used for cloning, sequencing, site-
directed mutagenesis, or in vitro transcription purposes.
• Phagemids are essentially plasmids that contain an origin of
replication for single-stranded phages (such as M13 or f1)
• Thus bacteria which are transformed with this plasmid and infected
with a helper phage (such as M13 or f1) can produce single-
stranded copies of plasmids, which in turn can be packaged into
phage heads.
• Since this vector is a hybrid of both plasmids and single-stranded
phage vectors, it can be used to generate single-stranded DNA to be
used in sequencing reactions.
Geckil

Geckil

Vectors for use in eukaryotic cells
• The genomes of eukaryotes are larger and more
complex than those of bacteria, so modifications of the
techniques are needed to handle the larger amounts of
DNA and the array of different cells and life cycles of
eukaryotes.
• For instance, some eukaryotic proteins cannot be easily
expressed in large amounts in bacteria, and eukaryotic
expression systems need to be employed.
• A widely used vector–expression system for eukaryotic
proteins is insect baculovirus, into which genes are
inserted and expressed at high rates in cultured insect
cells
Geckil

Geckil
Baculovirus is a very large
DNA virus (about 150 kb)
that infects insect cells.
To express a foreign gene
in baculovirus, the gene
of interest is cloned in
place of the viral coat-
protein gene in a plasmid
carrying a small part of
the viral genome.
The recombinant plasmid
is cotransfected into
insect cells with wild-type
baculovirus DNA for
high expression of the
foreign protein.

Artificial chromosomes
Geckil

Bacterial Artificial Chromosomes
• Large genome sequencing projects often require the
cloning of much longer DNA segments than cannot be
incorporated into standard plasmid cloning vectors.
• To meet this need, plasmid vectors have been
developed with special features that allow the cloning
of very long segments (typically 100,000 to 300,000 bp)
of DNA.
• Once such large segments of cloned DNA have been
added, these are large enough to be thought of as
chromosomes, and are known as bacterial artificial
chromosomes, or BACs.
Geckil

• BACs are based on F plasmids which help even
distribution (aka. plasmid partitioning) of these
large-sized recombinant plasmids to the next
generation cells.
• A typical BAC vector contains ori and rep
sequences to ensure replication of the vector and
the copy number, and par sequences for even
partitioning of the DNA to daughter cells.
• Additionally selectable (e.g., amp) and screenable
(e.g., lacZ) markers and phage promoters such as
the T7 promoter are required.
Geckil

Geckil
Bacterial artificial chromosomes (BACs) as cloning vectors. After treatment with an
appropriate restriction endonuclease, a BAC and a long fragment of DNA are ligated. The
recombinant BAC is transferred into E. coli by electroporation, and colonies with
recombinant BACs are selected by growth on media containing both the antibiotic
chloramphenicol and X-gal, the substrate for –galactosidase that produces a colored
product.

Yeast Artificial Chromosomes
• As with E. coli, yeast genetics is a well-developed discipline.
• The genome of Saccharomyces cerevisiae contains only 14
million bp (about four times the size of the E. coli
chromosome), and its entire sequence is known.
• Plasmid vectors have been constructed for yeast,
employing the same principles that govern the use of E. coli
vectors.
• Some recombinant plasmids incorporate multiple
replication origins and other elements that allow them to
be used in more than one species (e.g., in yeast and E. coli).
Plasmids that can be propagated in cells of two or more
species are called shuttle vectors.
Geckil

• Yeast artificial chromosomes, or YACs contain all the
elements needed to maintain a eukaryotic chromosome in
the yeast nucleus:
– a yeast origin of replication
– two selectable markers
– specialized sequences (derived from the telomeres and
centromere) needed for stability and proper segregation of the
chromosomes at cell division.
• In preparation for its use in cloning, the vector is
propagated as a circular bacterial plasmid. Cleavage with a
restriction endonuclease removes a length of DNA between
two telomere sequences (TEL), leaving the telomeres at the
ends of the linearized DNA. Cleavage at another internal
site divides the vector into two DNA segments, referred to
as vector arms, each with a different selectable marker.
Geckil

• The genomic DNA to be cloned is prepared by partial digestion to obtain a
suitable fragment size.
• Genomic fragments are then separated by pulsed field gel
electrophoresis, a variation of gel electrophoresis that segregates very
large DNA segments.
• DNA fragments of appropriate size (up to about 2 million bp) are mixed
with the prepared vector arms and ligated.
• The ligation mixture is then used to transform yeast cells (pretreated to
partially degrade their cell walls) with these very large DNA molecules—
which now have the structure and size to be considered yeast
chromosomes.
• Culture on a medium that requires the presence of both selectable marker
genes ensures the growth of only those yeast cells that contain an artificial
chromosome with a large insert sandwiched between the two vector
arms.
Geckil

Geckil
Construction of a yeast artificial chromosome (YAC). A YAC vector includes an origin of replication
(ori), a centromere (CEN), two telomeres (TEL), and selectable markers (here designated X and Y). Two
separate DNA arms are generated by digestion with BamHI and EcoRI, each arm having a telomeric
end and one selectable marker. A large DNA fragment, produced by EcoRI digestion, is ligated to the
two arms, creating a YAC. The YAC is transferred into yeast cells (which have been prepared by
removing the cell wall to form spheroplasts). The transformed cells are selected for X and Y, and the
surviving cells propagate the DNA insert.

• One arm contains an autonomous replication sequence (ARS),
a centromere (CEN) and aselectable marker (trp1). The other
arm contains a second selectable marker (ura3).
• Insertion of DNA into the cloning site inactivates a mutant
expressed in the vector DNA and red yeast colonies appear.
• Transformants are identified as those red colonies which grow
in a yeast cell that is mutant for trp1 andura3. This ensures
that the cell has received an artificial chromosome with both
telomeres (because of complementation of the two mutants)
and the artificial chromosome contains insert DNA (because
the cell is red).
Geckil

Getting DNA into cells
In bacteria, the passage of genetic material happens
in three ways:
1. Conjugation - transfer using sex pili
2. Transduction - transfer by bacteriophages
3. Transformation - uptake of naked DNA from outside
the cell (In mammalian cell culture, the analogous
process of introducing DNA into cells is commonly
termed transfection)
Some of the other artificial methods include protoplast
fusion, microinjection , and the use of "gene guns" to
blast DNA-coated particles through cell walls and into
plant cells.
Geckil

Selection, screening, and analysis of recombinants
Geckil

• The lac Z fragment,
induced by IPTG, is
capable of intra-allelic
complementation with a
defective form of β-
galactosidase enzyme
encoded by host
chromosome.
• The complementation
results an active enzyme
which hydrolyses X-gal
(5-bromo-4-chloro-3-
indolyl- beta-D-
galactopyranoside) and
form blue colonies.
Geckil
The molecular mechanism involved for screening
recombinant cells

• Insertion of foreign DNA into the MCS located within the lac Z gene
causes insertional inactivation of this gene and abolishes intra-allelic
complementation.
• Thus bacteria carrying recombinant plasmids in the MCS cannot hydrolyse X-gal,
giving rise to white colonies contrary to non-recombinant cells which are blue.
Geckil
Principle of
blue/white
selection for
the detection of
recombinant
vectors

Geckil

Geckil
Alpha complementation
• Plasmid encodes N-terminus of
beta galactosidase (alpha
fragment)
• Host strain encodes the C-
terminus of beta galactosidase
(omega fragment)
• Beta galactosidase function is
only seen in the presence of both
the N- and C-terminal fragments
• Beta gal function can be
monitored by the cleavage of X-gal
which yields a bright blue product
(blue colonies on a plate)
Bright blue
X-gal

Geckil
• Plasmid encodes N-terminus of beta galactosidase (alpha
fragment), with an MCS
• Foreign DNA in the MCS, no alpha fragment
• No alpha fragment, no B-gal
• No B-gal, no blue color (white colonies)
pUC19
transformation
plate
Colony without foreign DNA in MCS
Colony with foreign DNA in MCS

Geckil
Gel electrophoresis is a laboratory
technique used to separate nucleic
acids of different sizes.
In gel electrophoresis, a porous gel is
often made from agarose (a
polysaccharide isolated from seaweed),
which is melted in a buffer solution and
poured into a plastic mold. As it cools,
the agarose solidifies, making a gel that
looks something like stiff gelatin.

Genetic Engineering: Cloning Vectors

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