SBT

3113

Cloning vectors

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CLONING VECTOR
=
An agent that can replicate in target cells and
the DNA segment that it carries
independently

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•  Has several properties:

o  Able to replicate itself and the DNA segment that it
carries independently
o  Relatively small
o  Contain different RE cleavage sites that are present only
once in the vector
o  Carry a selectable marker
o  Easy to recover from host cells

•  e.g of vectors: pUC18 (plasmid), lambda phage

(bacteriophage) and cosmid

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Plasmid vectors

•  Circular molecules of DNA that lead an independent

existence in bacterial cell

•  Normally carry one or more genes

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Plasmid vectors

•  Most widely used, versatile and easily manipulated

•  Naturally occurring extrachromosomal DNA molecules, usually

circular, double-stranded and supercoiled

•  Vary in size but plasmid used for gene cloning are usually
small, 2 – 5kb

•  Most of the commonly used ones are based on (or closely

related to) a naturally occurring E. coli plasmid called ColE1

•  Have the ability to disseminate antibiotic resistance genes

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 pBR322

•  The first genetically engineered plasmids to be used in

recombinant DNA technology
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pUC8 pUC18

pGEM3Z
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•  Plasmid replication

•  In genetic engineering, we make use of this ability of
plasmid to be replicated, as it enables us to insert pieces of
DNA which are then copied as part of the plasmid and hence
passed on to the progeny when cell replicates

•  The most fundamental property of a plasmid as a cloning

vector, is it ability to replicate in the host bacterium

•  Most or all the enzymes and other products needed for

replication are already present in the host, the amount of
information that the plasmid has to supply maybe only a
few hundred base pairs
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•  This region of the plasmid that is necessary for replication

is generally referred to as origin of replication, usually
designated ori in plasmid maps

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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York:
W. H. Freeman; 2000. Section 7.1, DNA Cloning with Plasmid Vectors. Available
from: http://www.ncbi.nlm.nih.gov/books/NBK21498/
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•  Plasmids that use the origin of replication from ColE1 or its

relatives are multi-copy plasmids. Wild type ColE1 is present
at about 15 copies/cell

•  Most of the engineered vectors used today are present as

many hundreds copies/cell. This makes easier to purify large
amounts of plasmid

•  Presence of so many copies can also be disadvantages.

Large amount of plasmid may make the host cell grow more
slowly. If the gene or its product in any way harmful or toxic
to the bacterium, it can sometimes be very difficult to
isolate the required clone

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•  Some plasmids are able to replicate in a wide variety of

bacterial species, but most that are used for cloning are
rather more restricted in their host range

•  It is possible to insert two origins of replication into the

plasmid so that it will be replicated in E. coli using one origin,
and in chosen host using alternative replication origin. Such
vector is called as shuttle plasmid

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Shuttle plasmid for transformation of Rhodococcus rhodnii
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•  Cloning sites

•  Is a unique restriction site, enzyme concerned will cut the

plasmid once only

•  A basic plasmid used as a cloning vector may contain only

one or two such unique restriction sites, which must located
in a region of the plasmid that is not essential for replication
or any other functions needed

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•  To facilitate cloning a multiple cloning site (MCS) ie a short

DNA region that contains recognition sites for a number of
different enzymes is inserted into the plasmid

Structure of plasmid Use of the plasmid cloning

cloning vector pUC18 vector pUC18
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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell
Biology. 4th edition. New York: W. H. Freeman; 2000.
Section 7.1, DNA Cloning with Plasmid Vectors.
Available from: http://www.ncbi.nlm.nih.gov/books/
NBK21498/ 20
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•  Selectable markers

•  Selectable markers are essential to circumvent the

problem of inefficiency of ligation and bacterial
transformation

•  Even with high efficiency systems that are available for E.

coli, the best yield available, using native DNA
transformation, implies that only about 1% of the bacterial
cells take up the DNA

•  Thus in order to be able to recover the transformed clones,

it is necessary to be able to prevent the non-transformed
cells (cells that have not taken up the plasmid)from growing

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•  The presence of an antibiotic resistance gene on the plasmid

vectors, allow us to plate out transformation mix on agar plate
containing relevant antibiotic and only the transformants will
be able to grow

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•  Insertional inactivation

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•  Insertional inactivation

•  In pGEM-T and pUC18 for example, the multiple cloning site

is located near the 5’ end of the β-galactosidase gene (lacZ’)

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•  The synthetic oligonucleotide that creates the MCS does

not affect the reading frame of the lacZ gene

•  pGEM-T and pUC18 carry a part of the lacZ gene, and E. coli
strains that carry the remainder of the gene are being used as
the host

•  The product of the host gene is unable to hydrolyse lactose

by itself, and so the host strain without the plasmid is Lac-, ie
does not ferment lactose

•  When pGEM-T or pUC18 is inserted into the host, the

plasmid-encoded polypeptide will associate with the host
product to form a functional enzyme. pGEM-T (or pUC18) is
said to complement the host defect in lacZ
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•  The activity of the β-galactosidase can be detected by

plating the organism onto agar plate containing the
chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-
galactopyranoside (X-gal) with inducing agent isopropyl
thiogalactoside (IPTG)

•  The X-gal substrate is colorless but the action of β-

galactosidase releases the dye moiety, resulting in deep blue
color

•  Colonies carrying pUC18 (or pGEM-T) are therefore blue

when grown on this medium

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Principles of blue-white screening

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•  Successful insertion of a DNA fragment at the cloning site

results in the gene being disrupted and the resulting E. coli will
be unable to cleave X-gal, resulting bacterial colonies refer as
white colonies

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Blue-white screening

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•  Advantage of plasmid vectors: small, easy to manipulate,

conceptually simple and universal and can be used for a wide
range or organisms

•  Disadvantage: limited in cloning capacity

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Bacterial transformation

•  After obtaining recombinant plasmids the next step is to

introduces them into E. coli (host)

•  This is called transformation and involves two steps:

i.  To get DNA into the bacterial cell

ii.  To select those cells which contain the plasmid


•  Transformation can be defined as the uptake of naked DNA

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Bacterial transformation

•  Was discovered in 1928, where pneumococcus has the

natural ability to take up ‘naked’ DNA from its surroundings.
This ability is known as competence

•  Competence develops naturally in some bacterial species but

not in some . For example E. coli does not seem to exhibit
natural competence

•  Purified DNA is actively or passively imported into host cells
using non-viral methods
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Griffith’s experiment of
bacterial transformation

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Bacterial transformation

•  Two methods for transforming bacteria:

i.  Chemical method utilising CaCl2 and heat shock

ii.  Electroporation – uses a short pulse of electric
charge to facilitate DNA uptake

•  The major difference between this two techniques is

transformation efficiency; electroporation is several orders
of magnitude more efficient than transformation by CaCl2

•  Transformation frequencies are generally quoted in

number of transformants per µg of DNA
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 Chemical transformation using CaCl2 and heat
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Selection of transformants
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•  For chemical treatment during transformation the DNA

associated with the lipopolysaccharide on the outer space of the
competent cells, uptake of this DNA is associated with damage to
the cell walls caused, in part, by the Ca2+ ions and with the heat
shock

•  For electroporation, it is thought that the high voltage pulse

makes the bacterial membrane more permeable and possibly
moves the DNA into the bacterium by a process akin to
electrophoresis

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Lambda bacteriophage
•  Lambda biology

•  Plasmid vectors are best to be used for relatively small
fragments

•  Recombinant plasmid may become rather less stable with

larger DNA inserts, the efficiency of transformation is
reduced and the plasmid will give a much smaller yield when
grown and purified in E. coli

•  Vectors based on bacteriophage lambda allow cloning of

larger fragments, which is important in constructing gene
libraries
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•  The larger the inserts, the fewer clones you have to screen to
find the one you want

•  For library construction, bacteriophage plaques are much

cleaner than those obtained with bacterial colonies

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 Two main types of phage structures:
(a) head-and-tail e.g λ and (b) filamentous e.g M13

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The general pattern
of infection of a
bacterial cell by
bacteriophage

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The life cycle of bacteriophage λ
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•  Lysogeny

•  Lambda is a temperate bacteriophage

•  On infection of E. coli it may enter a productive lytic cycle,

resulting in lysis of the cell and releases a number of phage
particles

•  Or it may enter a more or less stable relationship with the

host known as lysogeny

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The lysogenic infection cycle of
bacteriophage λ

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•  Some phage mutants will only produce lytic infection and
these give rise to clear plaques, while the wild type phage
produces turbid plaques due to the presence of lysogens
which are resistant to further attack by lambda phage (known
as superinfection immunity)

•  Some bacterial strains carrying a mutation known as hfl

(high frequency of lysogenization) produce a much higher
proportion of lysogens when infected with wild-type lambda

•  When using lambda vectors, lytic cycle is the more relevant

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•  In the lysogenic state, lambda is normally integrated into
the bacterial chromosome, by site-specific recombination at
a specific position and is replicated as part of the bacterial
DNA. The newly integrated genetic material is called
prophage

•  Induction of the lysogen requires excision from the

chromosome. However the integration is not essential
feature of lysogeny. Lambda can continue to replicate in an
extrachromosomal, plasmid-like state

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•  Also, expression of almost all of the phage genes is
switched off by the action of a phage-encoded repressor
protein, encodes by the cI gene. The expression of this gene
requires two other genes, cII and cIII

•  Particles of a wild-type bacteriophage lambda have a

double-stranded linear DNA genome of 48 kb, with 12 bases
at each end are unpaired but complementary

•  These ends are ‘sticky’ or ‘cohesive’, the longer length of the

sticky ends makes the pairing much more stable

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•  When lambda infects a bacterial cells and injects its DNA into
the cell, it will form a circular structure, with the nicks being
repaired in vivo by bacterial DNA ligase

•  At about this time, a complex series of events occur that

affect subsequent gene expression, determining whether the
phage enters the lytic or lysogeny cycle

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•  Lytic cycle

•  Circular DNA is initially replicated, in a plasmid-like manner
(theta replication) to produce more circular DNA

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•  Eventually, replication switches to an alternative mode
(rolling-circle replication) which generates a long linear DNA
molecule containing a large number of copies of the lambda
genome joined end to end in a continuous structure

•  The genes carried by the phage are being expressed to

produce the components of the phage particle

•  These proteins are assembled into two separate

structures: the head and the tail

•  The packaging process involves enzymes recognizing

specific sites on the multiple-length DNA molecule and
making asymmetric cuts in the DNA
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•  These staggered breaks in the DNA give rise to the cohesive
ends; these sites are called cohesive end sites (cos sites)

•  Accompanying these cleavages, the region of DNA between

the two cos sites (representing a unit length of the lambda
genome) is wound tightly into the phage head. This process is
known as packaging

•  Following successful packaging of the DNA into the phage

head, the tail is added to produce the mature phage particles
which eventually releases when the cell lyses

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•  Some of the DNA can be deleted. This is possible because the

lambda genome contains a number of genes that are not
absolutely necessary

•  The ‘non-essential’ region contains most of the genes involved in

integration and excision of the λ prophage from E. coli
chromosome

•  A deleted λ genome is therefore non-lysogenic and can follow

only the lytic infection cycle

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The λ genetic map showing the important genes
and the functions of the gene clusters

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 The λ genetic map showing the position of the main non-essential
region that can be deleted without affecting the ability of phage to
follow the lytic infection cycle

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•  However the stability of the phage head requires a certain

amount of DNA. To produce viable phage, there has to be a
minimum of 37 kb of DNA between the two cos sites that are
cleaved

•  The existence of these packaging limits is a very important

feature for designing and application of lambda vectors

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 Using natural selection
to isolate λ phage
lacking EcoRI restriction
sites

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•  Lysis of the bacterial cell is accomplished primarily through the

action of phage-encoded protein, encodes by the gene S.
Mutations in this gene can cause a delay or failure of lysis – can
lead to the increase in the yield of bacteriophage. Many lambda
vectors have this mutation

•  One of the most important feature to consider when using

lambda vectors is the length of DNA that will be packaged into the
head, as it is determined by the distance between two cos sites

•  If a piece of DNA is inserted into lambda vector, the distance will

be increased and the amount of DNA to be packaged will be
bigger

•  The head size is fixed so it can only accommodate a certain

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Examples of λ cloning vectors

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•  Insertion vectors

•  The simplest form of lambda vector is known as an
insertion vector, contains a single cloning site which DNA
can be inserted

•  Wild-type lambda DNA contains many sites for the most

commonly used restriction enzymes, so all lambda vectors
have been genetically manipulated to remove unwanted
restriction sites

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•  One example of a lambda vector is gt10, where there is only
a single site for EcoRI to cut the DNA and a reduced size of
the phage DNA (43.3 kb)

•  Cutting this vector with EcoRI will produce two fragments;

the left and the right arms

•  One end of each of the two fragments is derived from the

cohesive ends of the lambda DNA, and will therefore anneal
quite stably at 37°C – so although not covalently joined they
can be considered as a single DNA fragment

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•  gt10 is also an example for insertional inactivation to be used

• The EcoRI site is found within the repressor (cI) gene, so the
recombinant phage (which carry an insert at this position), are
unable to make functional repressor. They will be unable to
establish lysogeny and will give rise to clear plaques (parental
phages will give turbid plaques)

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•  Another example of insertional vector is lambda gt11,
where it contains a β-galactosidase gene and has a single
EcoRI site within that gene and the cloning site is towards
the 3’ end of the β-galactosidase gene

•  Insertion of DNA at the EcoRI site will inactivate the β-

galactosidase gene, so the recombinants will produce
colourless plaques on a medium containing X-gal

•  If the insert is in the correct orientation and in frame, will

give rise to a fusion protein containing the product
encoded the insert fused to β-galactosidase protein

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•  Lambda gt11 is an example of translational fusion protein
vector

•  The packaging limits for lambda DNA are between 37 kb
and 51 kb. The maximum cloning capacity for an insertion
vector is 14 kb. In order to increase the available cloning
capacity, another lambda vector known as replacement
vector can be used

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•  Replacement vectors

•  To circumvent the problem of packaging limits, an
alternative lambda vector was designed

•  This vector is called replacement vector because the piece

of DNA can be removed and replaced by the insert

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•  There are two sites where DNA can be cleaved, resulting in three
fragments; the left and right arms (which will anneal because of
the presence of the cohesive ends) and a third fragment which is
not needed (except to maintain the size of the DNA) and can be
discarded. This third fragment is called a stuffer fragment

•  One example of replacement vector is EMBL4. The cloning

capacity of the vector is up to 22 kb

•  In EMBL4, the combined size of the arms is only 29 kb, which is

less than the minimum requirement for packaging

•  Any arms that are ligated minus the insert will be too small to
produce viable phage particles. Viable particles will only be
produced if ligation result in an insert at least 8 kb. This provide
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Different strategies for cloning with a λ vector
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Cosmids
•  Cosmids provide larger cloning capacity compared to lambda replacement
vectors

•  Is a plasmid which contains a cos site. It has an origin of replication, a

selectable marker and a cloning site

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•  Digestion, ligation with the insert and subsequent purification of recombinant

clones are carried out as for a normal plasmid vector

•  However, instead of transforming bacterial cells with the ligation mix, the
ligation mixture is subjected to in vitro packaging. Since the cosmid is quite
small, the packaging reaction will only be successful if DNA fragment between
32 – 45 kb is being inserted

•  The products of the packaging reaction although are phage particles, will not
give rise to more phages after infection of a host bacterium

•  The cosmid will replicate as a plasmid and will give rise to more cosmid-
containing cells, thus can be selected as colonies on agar containing an
antibiotic

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•  Cosmids have been used for genome mapping and sequencing projects for
example bacterial genome

•  Cosmids have some advantages over phage lambda:

§  Can be propagated and purified by conventional plasmid-oriented
techniques

§  The vector is small compared with the insert

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M13 vectors
•  M13 is a bacteriophage that infects E. coli

•  It is a ‘sex-specific’ ie F-specific bacteriophage. It attaches to the tips of the

pili that are produced on the surface of bacteria that carry an F-type plasmid

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•  M13 has a very long, thin filamentous phage particles containing a circular,
single stranded DNA molecule with the size of 6 kb

•  The smaller size of the M13 DNA molecule means that it has fewer genes
than the λ genome

•  M13 capsid is constructed from multiple copies of just three proteins

(requiring only three genes)

•  M13 follows a simpler infection cycle than λ and does not need genes for
insertion into the host genome

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•  After DNA enters the cell, it is converted to a double-stranded molecule (the
replication form, RF) by synthesis of the complementary strand

•  This molecule is replicated by producing a circular single-stranded copy of

one strand of the RF. This single-stranded DNA is again converted to a double-
stranded form

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Infection cycle of bacteriophage M13

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•  The production of the single-stranded intermediate requires a specific signal

on the DNA, and is therefore completely strand-specific

•  Separation of the synthesis of the two strands is not only unique to M13 but
can be found in other bacteriophages and some classes of plasmids

•  However most of the phage DNA within the cell is double-stranded circular
DNA (RF) and can be isolated by conventional plasmid purification methods

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•  Continued replication of the phage leads to a build up of these plasmid-like

DNA molecules within the cell. At the same time, expression of phage genes
occurs and the product of one of these genes binds to the single-stranded
product, initiating production of phage particles

•  This occurs by extrusion of DNA through the cell membrane during which
process it becomes coated with phage proteins molecule

•  The length of the filamentous phage particle is determined by the length of

the DNA thus there are no absolute packaging limits for M13

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•  Significant feature of M13 is that infection does not lead to bacterial lysis.
Phage particles continue to be produced and the cells remain viable

•  Infection does result in the appearance of ‘plaques’ in a bacterial lawn (zones

of reduced growth)

•  Main advantage of M13 vector is that it provides a very convenient way of

obtaining single-stranded versions of a gene used for DNA sequencing and
site-directed mutagenesis

•  Vectors based on M13 have been engineered to contain MCS and usually
include a β-galactosidase gene to distinguish recombinants from parental
vector

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Bacteriophage M13 genome

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Bacteriophage M13 vector 105
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Phagemid
•  Although M13 vectors are useful for the production of single-stranded
versions of cloned genes, they have one disadvantage

•  There is a limit to the size of DNA fragment that can be cloned with a M13
vector, with 1.5 kb as maximum capacity, though 3 kb fragments have been
occassionally cloned

•  To solve this problem a hybrid vector, phagemid has been developed,

combining part of M13 genome with plasmid DNA

•  Example is pEMBL8, made by transferring 1.3 kb fragment of M13 genome

into pUC8. This fragment contains the signal sequence recognized by the
enzymes that convert normal double-stranded M13 molecule into single-
stranded DNA before secretion of new phage particles

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•  pEMBL8 molecules are also converted into single-stranded DNA and

secreted as defective phage particles

•  E. coli cells necessary as hosts for pEMBL8 cloning experiments are

subsequently infected with normal M13 to act as a helper phage, providing
the necessary replicative enzymes and phage coat proteins

•  pEMBL8 has the polylinker cloning sites within the lacZ’ gene, so
recombinant plaques can be identified on agar containing X-gal

•  Using pEMBL8, single-stranded versions of cloned DNA fragments up to 10

kb in length can be obtained

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Transfection

•  Equivalent to transformation but phage DNA rather than plasmid is involved

•  Purified phage DNA or recombinant phage molecule is mixed with

competent E. coli cells and DNA uptake induced by heat shock

•  Is a standard method for introducing the double-stranded RF form of a M13

vector into E. coli

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•  Selection after transfection can be done by mixing the transfection mix with a
culture of a phage-sensitive bacterium in molten soft agar and look for plaques
(zone of clearing due to lysis of the bacteria) when overlaid onto an agar plate

•  The large size of most bacteriophage makes transfection an inefficient
process. However there is an efficient alternative

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In vitro packaging

•  Some mutant lambda phages, in an appropriate bacterial host strain will
produce empty phage heads, while others are defective in the production
of the head, but contain the proteins needed for packaging

•  Use of the mixture allows packaging of added DNA, which occurs

effectively in vitro

•  This packaging reaction is most efficient with multiple length DNA. The
enzyme involved in packaging the DNA normally cuts the DNA at two
different cos sites on a multiple-length molecules

•  For lambda vectors it is advantageous to adjust the ligation conditions so

that multiple end-to-end ligation of lambda molecules together with the
inserts can be achieved

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Packaging of bacteriophage DNA

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•  It is possible to package DNA into virus particles in the test tube and
then use to infect E. coli; referred as in vitro packaging and can yield about
109 plaques

•  Mixing recombinant DNA with viral head precursors, phage tails and
other proteins required for packaging results in mature viral particles
containing recombinant DNA, which can be used to infect E. coli cells and
which will produce plaques on a bacterial lawn in the usual way

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•  Single strain system:

§  Defective λ phage carries mutation in the cos sites; so these are not
recognized by the endonuclease that normally cleaves the λ catenanes
during replication
§  The defective phage cannot replicate, though does direct synthesis of all
proteins needed for packaging
§  The proteins accumulate in the bacterium and can be purified from
cultures of E. coli infected with the mutated λ and used for in vitro
packaging of recombinant λ molecules

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•  Two defective λ strains system:

§  Two defective strains are needed, each strain carry a mutation in a gene for
one of the components of the phage protein coat; one mutation in gene D
and the other in gene E
§  Neither strain able to complete an infection cycle in E. coli because in the
absence of the product of the mutated gene the complete capsid cannot be
made. Instead the products of all the other coat protein genes accumulate
§  An in vitro packaging mix can therefore be prepared by combining lysates
of two cultures of cells, one infected with λ D- strain, other with E- strain.
The mixture now contains all the necessary components for in vitro
packaging

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•  With both systems, formation of phage particles is achieved
simply by mixing the packaging proteins with λ DNA – assembly
of the particles occurs automatically in the test tube

•  The packaged λ DNA is then introduced into E. coli cells simply by

adding the assembled phages to the bacterial culture and
allowing the normal λ infective process to take place

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Identification of recombinant phages

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Supervectors

i)  YAC

•  Although cosmids were the first vectors used to develop mammalian

gene libraries, their limited capacity still not suffice for human genome
project

•  A supervector that is able to carry 100 kb or more was developed and one
example is the yeast artificial chromosome (YAC)

•  As with the shuttle vector, the YAC vector is propagated as a circular

plasmid in E. coli

•  Restriction enzyme digestion removes the stuffer fragment between the

two telomeres and cuts the remaining vector into two linear arms, each
carrying a selectable marker
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Chromosome structure

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•  The insert is then ligated between these arms as in the case of phage
lambda and transformed into a yeast cell, with selection for
complementation of both auxotrophic marker

•  YACs are usually used to clone 600 kb fragments, and specialised vectors
can accommodate up to 2Mb insert

•  For routine library construction insert sizes are usually in 250 to 400 kb
range

•  However YACs have problem with stability on the insert, especially large
fragments which can be subjected to rearrangement by recombination.
The recombinant molecules are not easy to recover and purify

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Host: yeast strain with double auxotrophic mutant, trp1- and
ura3-
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ii) BAC

•  Larger bacterial vectors are used more than YACs even though their
capacity is lower

•  For example vectors based on bacteriophage P1, which can accommodate

100 kb insert and bacterial artificial chromosomes (BACs) which are based
on the F plasmid and can accommodate 300 kb insert

•  These vectors lack the instability problems found in YACs and play an
important role in genome sequencing projects

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The main features of a typical bacterial artificial chromosome
(BAC)

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Expression vectors

•  Inclusion of transcriptional and translational signals into a vector resulted in

the formation of an expression vector

•  There are two main types:

•  If the vector just carries the promoter, and relies on the translational
signal present in the cloned DNA, it is referred as transcriptional fusion
vector

•  If the vector also supplies the signals, it is called translational fusion

vector

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Expression vectors: transcriptional fusion

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Expression vectors: translational fusion

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•  For expression vector, we need to be able to control the expression, whether

to have the expression on or off

•  One example of promoter that can be used to control expression is the T7
promoter, from bacteriophage

•  In T7, this promoter controls the expression of the ‘late’ genes ie genes that
are only switched on at a late stage of infection

•  T7 promoter is not recognised by E. coli RNA polymerase but requires the T7

RNA polymerase. So if DNA fragment is cloned downstream of the T7 promoter
using an ‘ordinary’ E. coli host, no expression can be achieved

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•  Once the right construct is obtained, the plasmid can be isolated and put
into another E. coli strain that has been engineered to contain a T7
polymerase gene

•  If the expression of the T7 polymerase is itself regulated, for example by

putting it under the control of lac promoter, then the expression of the T7
polymerase can be turned up (by adding IPTG) and down

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Causey/pET.html 138
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Vectors for cloning and expression in eukaryotic

cells

•  Most primary cloning is carried out using bacterial host because ease of
manipulation and range of powerful techniques established

•  Eukaryotic hosts are commonly used to study the behaviour of genes that
have already been cloned; for analysing their effect on the host cell and
modifying it or for obtaining a product which is not made in its natural state in
bacterial host

•  The eukaryotic vectors are primarily in obtaining gene expression; not for
gene libraries or primary cloning

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•  Yeasts

•  Commonly used yeast for expression is Saccharomyces cerevisiae.
Another example is Pichia sp.

•  The vector primarily being used is yeast episomal plasmids (YEp). This
vector able to replicate independently in yeast at high copy number
(25-100 copies/cell). This plasmid also have E. coli origin of replication
thus able to be grown and manipulated in E. coli host (shuttle vectors)

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Structure of yeast episomal vector

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YEp13

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•  For selectable marker, there are fewer antibiotics available which yeasts
are sensitive. Therefore selection are more commonly made using
complementation of auxotrophic mutations in the host strain

•  For example, a host strain of S. cerevisiae with a mutation in the trp1

gene will be unable to grow on a medium lacking tryptophan. If the
vector plasmid carries a functional trp1 gene, then the transformants can
be selected on a tryptophan-deficient medium

•  Other commonly used selectable markers include ura3, leu2 and his3

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Using LEU gene as a selectable marker in yeast cloning
experiment
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•  Since one of the purpose of using yeast as a host is to express the cloned
gene, these vectors are commonly designed as expression vectors

•  Expression signals are included in the vector and sometimes with other
signals (secretion or targeting to the nucleus or other compartments)

•  Although S. cerevisiae is an eukaryote, it has few introns, so is not the

host of choice if we want to ensure correct excision of introns

•  Other example of yeast vectors: yeast integrating plasmid (YIp) and

yeast replicative plasmids (YRps)

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•  Insects

•  Due to importance of Drosophila melanogaster, vectors for cloning
genes in this organism are available

•  Cloning in Drosophila makes use of a transposon called the P element

•  Transposons are common in all types of organism. They are short pieces
of DNA (usually less than 10 kb) that can move from one position to
another in the chromosomes of a cell

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•  P elements which are one of several types of transposon in Drosophila,
are 2.9 kb and contain 3 genes flanked by short inverted repeat sequences
at either end of the element

•  The genes code for transposase, the enzyme that carries out the
transposition process and the inverted repeats form the recognition
sequences that enable the enzyme to identify the two ends of the inserted
transposon

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•  As well as moving from one site to another within a single chromosome, P
elements can also jump between chromosomes, or between a plasmid
carrying a P element and one of the fly’s chromosome

•  The latter is the key to the use of P elements as cloning vectors

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•  The vector is a plasmid that carries two P elements, one of which contains the
insertion site for the DNA that will be cloned

•  Insertion of the new DNA into this P element results in disruption of its
transposase gene, so this element is inactive

•  The second P element has an intact version of transposase gene. Ideally this
second element should not itself be transferred to the Drosophila chromosomes,
so it has its ‘wings clipped’; its inverted repeats are removed so that the
transposase does not recognize it as being a real P element

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•  Once the gene to be cloned has been inserted into the vector, the
plasmid DNA is microinjected into fruit fly embryos

•  The transposase from the wings-clipped P element directs transfer of

the engineered P element into one of the fruit fly chromosomes

•  If this happens within a germline nucleus, then the adult fly that
develops from the embryo will carry copies of the cloned gene in all its
cells

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•  Mammalian cells

•  With cloning in mammalian cells, independent, plasmid-like replication
is often not sustained

•  Some vectors are capable of plasmid-like replication, especially those

carrying the origin of replication from the virus SV40 (simian virus 40),
which replicate episomally in some mammalian cells (for example COS
cells)

•  More stable clones are obtained by inserting the DNA into the
chromosome, which happens readily in mammalian cells

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Structure of a basic episomal vector for gene expression in

mammalian cells
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•  SV40 is capable of infecting several mammalian species, following a lytic

cycle in some hosts and a lysogenic cycle in others

•  The genome is 5.2 kb in size and contains two sets of genes, the ‘early’
genes (expressed early in the infection cycle and coding for proteins
involved in viral DNA replication and the ‘late’ genes, coding for viral capsid
proteins

•  Cloning with SV40 involves replacing one or more of the existing genes with
the DNA to be cloned

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References:

i.  Dale, J. W., von Schantz, M. and Plant, N. (2012). From genes
to genomes. Wiley-Blackwell.

ii.  Lodge, J., Lund, P. and Minchin, S. ( 2007). Gene cloning:

Principles and applications. Taylor and Francis.

iii.  Brown, T. A. (2010). Gene cloning and DNA analysis: An

introduction. Blackwell Publishing.

iv.  Howe, C. (2007). Gene cloning and manipulation. Cambridge

Press.

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