PSB SCIE3326

VECTORS FOR LARGE

INSERTS, EXPRESSION &
SHUTTLE VECTORS
Prof. Barbara Chang
[email protected]

Microbiology and Immunology,

School of Pathology & Laboratory Medicine
The University of Western Australia
 Learning Objectives
• At the end of this lecture, you will be able
to describe, using diagrams, the features,
uses and examples of:
Phage vectors
Cosmid vectors
Expression vectors
Shuttle vectors
Artificial chromosomes
References:
1. Lodish H. et al. (2004) “Molecular Cell Biology” 5th Ed. Chapter 9, Chapter 10 p 437.
2. Sambrook and Russell (2001) “Molecular Cloning: A laboratory manual” 3rd Ed.
Chapters 4, 15.
3. Griffiths A.J.F. et al. (2005) “Introduction to Genetic Analysis” 8th Ed. Chapter 11.
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 PHAGES AS VECTORS
• Can accommodate more foreign DNA
than plasmids e.g. 15 kb or more
 Useful for constructing gene libraries
• Phages infect cells more efficiently
than plasmids transform cells, so yield
of clones higher
plaque: hole/clearing in
• Clones are not colonies, but plaques bacterial lawn
 Clone detection by hybridisation is easier
• Minimum size requirement for inserts
 Advantage as gene library does not waste
space on clones with small inserts
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Bacteriophage  is a temperate, linear DNA phage
(48.5 kb) with a lytic and a lysogenic life cycle.

7 6 Synthesis of
Cell lysis and release viral particles
of progeny phage
100 phage/cell
Lytic mode

5
Replication of ~50 copies/cell
phage DNA
1 2
Lysogenic mode

3
9
Integration of phage
Induction
DNA into host DNA
4 8
UV radiation
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5
 LAMBDA REPLACEMENT
VECTORS
• Genes essential for lytic pathway are kept
• Middle region of genome not essential, replaced
with foreign DNA (hence the name)
• cos sites are needed for packaging DNA into
phage head
• Distance between cos sites must be 40-50 kb to
be packaged
• Only DNA with inserts between ~10-20 kb will be
packaged (insert DNA plus remaining phage DNA = 40-50 kb)
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• “Stuffer” fragment is cut out by RE leaving two “arms”
• Foreign DNA ligated to the arms
• Construct is packaged into lambda heads, using
packaging mix (commercially available)

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After cloning, how do you find which
phages have taken up an insert i.e. which
are recombinant?
In order to differentiate between wild-type and
recombinant phage, genes are placed onto the
stuffer fragment whose loss provides a detectable
phenotype
a) LacZ loss : blue-white screening
• lacZ inserted onto stuffer part of vector
• Loss of lacZ monitored by blue/white colour
of plaques on X-gal plates

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gt11 vector: an example of a phage vector
for which blue-white screening is used
• CLONING CAPACITY: 7.2 kb
 gt11 vector
• CLONING SITE:
– Unique EcoR I site 53 base
pairs upstream from b-
galactosidase termination
codon
• SELECTION:
– Plate transfected E. coli onto
agar containing X-gal and
IPTG.
– White plaques contain
vector plus insert, as b-
galactosidase gene is
disrupted and no enzyme is
produced www.bch.msu.edu/bchug/
web/bch472/472lm23.htm 9
b) Spi phenotype
• stuffer fragment carries phage genes red and gam, involved
in but not essential for lambda replication (red- and gam- phage
mutants replicate at slower rate in E. coli)
• E. coli host used in cloning is E. coli (P2) lysogen (P2 is a
temperate phage: this strain of E. coli carries P2 as a prophage)
• Lambda vector cannot form plaques on E. coli (P2) due to
presence of red and gam genes
 called Spi phenotype, for sensitive to P2
prophage inhibition
• Recombinant lambda phage vector loses red and gam, thus
forms plaques. All plaques contain insert!
Once the DNA is packaged into the phage, it is
introduced into the host via transfection

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A popular  replacement vector for construction of libraries
using Spi phenotype is EMBL3A with SalI, BamH1 and
EcoRI sites flanking the stuffer fragment.
It’s a partial
digest, so not
all RE sites
are cut, thus
overlapping
fragments are
produced

The BamH1
and Sau3A
ends ligate

Each plaque contains a vector plus insert; they are all recombinants
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 COSMID VECTORS
• are small (~5 kb) plasmids
• behave both as plasmids and as phages
• do not occur naturally, they are constructed
vectors which contain:
 a bacterial ori (usually
ColE1)
 a selective marker
(e.g. Ampr)
 a MCS or other cloning
site
 the 12 bp cos site of
lambda phage
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Role of cos sites in packaging
• when wild-type lambda DNA replicates, it forms multiple
copies of itself attached in one long piece called a
concatemer. These are cleaved into 48 kb pieces at the cos
sites to be packaged into phage heads. The lambda DNA
must be about this size to be packaged
• so when a DNA fragment of ~35-45 kb is cloned into a 5
kb cosmid, the recombinant DNA is the right size for
packaging
• the target DNA must be cut into this size range using
partial RE digestion e.g. Sau3A

• newer cosmids have 2 cos sites and do not require this

step
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COSMID CLONING

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 COSMID CLONING
 digest cosmid DNA with RE e.g. BamHI
 partially digest target DNA e.g. with Sau3A
 produces sticky ends which are compatible with BamHI ends
 select 35-45 kb fragments
 ligate to produce a linear concatemer of vector-insert-vector etc.,
which has multiple cos sites
 package DNA into lambda heads
 packaging extracts made using extracts from bacteria infected
with mutant strains of lambda can be commercial, or “home-
made”
 they contain all the components necessary for the insertion of
DNA into the phage
 infect E. coli host with phage, select Tetr colonies
 cosmid re-anneals to form circular plasmid
 cosmid does not have phage lytic genes so does not kill the host

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 USE OF COSMID VECTORS
• high capacity vectors suitable for construction
of genomic libraries
• longer inserts reduce the number of clones
required (compared to plasmid or phage vectors)
• N = [ln(1-P)]/[ln(1-f)]
• e.g. library of 750,000 cosmids (40 kb inserts) that
covers the human genome 10 times, gives 99.995%
probability of finding any one gene of interest

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 EXPRESSION VECTORS
Designed to yield the protein product of a cloned
gene, usually in the greatest amount possible
Include strong bacterial promoters and bacterial
ribosome binding sites
It is usually advantageous to keep a cloned gene
repressed until it is time to express it
• eukaryotic proteins produced in large amounts in
bacteria can be toxic
• cloned protein may form insoluble aggregates called
inclusion bodies

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Expression vectors:
Features
 Promoters for controlled
expression
 Bacteriophage e.g. T7,
SP6 (see diagram)
 Bacterial e.g. Plac
 Eukaryotic
 Ribosome binding site
 Shine Dalgarno
 Kozac
 Examples
 pGEM – in vivo and in
vitro protein expression
 Gateway – in vivo protein
expression in any host
Promega Corp. 18
Cloning into Expression vectors:
in vivo protein expression from pGEM

mRNA

ATG Stop
T7 phage Shine
promoter Dalgarno
Sequence

Transcription induction from this

promoter will only occur in an E. coli host
strain containing the gene encoding the
T7 phage RNA polymerase (e.g.
BL21(λDE3) ). 19
 Cloning into Expression vectors:
in vitro protein expression from pGEM
 Cell free transcription systems
 contain ribonucleotide
triphosphates, a buffer system
that includes dithiothreitol (DTT)
and Mg2+, and an appropriate
phage RNA polymerase
 Cell free translation systems
Rabbit Reticulocyte Lysate

In vitro transcription and translation


 treated with micrococcal
nuclease to destroy endogenous
mRNA
 contains all the cellular
components necessary for
protein synthesis (tRNA,
ribosomes, amino acids, and
initiation, elongation and
termination factors).
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 SHUTTLE VECTORS
A cloning vector that can replicate in two or
more host organisms. Needed for:
1. Cloning in different bacterial genera: e.g. pUC ori’s only
function in E. coli cells
2. Expression of cloned eukaryotic genes:
• E. coli may degrade the protein product of a cloned
eukaryotic gene
• prokaryotes may not carry out post-translational modifications e.g.
addition of sugar molecules
• frequently, the protein is incorrectly folded and may be inactive or
form inclusion bodies

Often the initial cloning is done in E. coli, and the

recombinant DNA is then transferred to the organism of choice.
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 One example of a class of such shuttle vectors is Yeast
Artificial Chromosomes (YACs). YACs replicate in E. coli
and in yeast (Saccharomyces cerevisiae)

The YAC contains for use in a

bacterial host (E. coli):
• an origin of replication (ori)
• a marker (ampR) for selection
based on ampicillin resistance

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 One example of a class of such shuttle vectors is Yeast
Artificial Chromosomes (YACs).

The YAC contains for use in a

yeast host:
• centromeric DNA (CEN4) and
• telomeres just like any
eukaryotic chromosome.
• an origin of replication, called
an autonomously replicating
sequence (ARS1).

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 One example of a class of such shuttle vectors is Yeast
Artificial Chromosomes (YACs).
The YAC also contains for use in a
yeast host:
• marker genes for selection in yeast cells
(TRP1 & URA3).
TRP1 is required for tryptophan
biosynthesis and URA3 is required
for uracil biosynthesis.
The YAC can then transform yeast
cells which cannot make tryptophan
or uracil, allowing them to grow on a
minimal agar plate.
The foreign DNA
can be inserted
into the YAC via
cleavage at the
EcoRI site and
then ligation.
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 Artificial chromosomes can carry
the largest DNA inserts
• Plasmid based
– Bacterial artificial chromosome
(BAC)
– Derived from F plasmids
– CLONING CAPACITY:120-300
kb, more stable than YACs
– Important in whole genome
sequencing e.g. human
genome, and in construction of
synthetic genomes
• Yeast based
– Yeast artificial chromosome
(YAC)
– CLONING CAPACITY: 100-
1000 kb
– Important as shuttle vectors
Griffiths 8th Ed Fig 11-9, 11-26
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