Lecture #02

CONSTRUCTION OF
CLONING VECTOR BY
λPHAGE

Visiting Lecturer
Fidaa Aslam
VECTORS

• There are two types of vectors

• Cloning vectors
• Expression vectors
 CLONING VECTORS
 A a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be
stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning
purposes.
 Used as a vehicle to artificially carry foreign genetic material into another cell
ESSENTIAL FEATURES OF A CLONING VECTOR
All commonly used cloning vectors have some essential features:
ORIGIN OF REPLICATION (ori):
 Makes autonomous replication in vector
 Ori is a specific sequence of nucleotide from where replication starts
CLONING SITE:
 Cloning site is a place where the vector DNA can be digested and desired DNA can be inserted by
the same restriction enzyme.
 Recently recombinant plasmids contain a multiple cloning site (MCS) which have many (up to ~20)
restriction sites.
SELECTABLE MARKER
 Is a gene that confers resistance to particular antibiotics or selective agent that would
normally kill the host cell or prevent its growth
 A cloning vector contains a selectable marker, which confer on the host cell an ability to
survive and proliferate in a selective growth medium containing the particular antibiotics
REPORTER GENE OR MARKER GENE
 Reporter genes are used in cloning vectors to facilitate the screening of successful clones by
using features of these genes that allow successful clone to be easily identified
 Such feature present in cloning vectors is used in blue-white selection
Additional Properties of Vectors:
 It should be short, small
 Compatible with host cell
 Should become high in copy number
 It should able to express itself utilizing the host machinery
What is copy number?
 Copy number refers to the number of molecules of an
individual plasmid that are normally found in a single
bacterial cell
 Some bacterial cells, especially large ones are stringent and
have a low copy number i.e. one or two per cell
 While others are relaxed plasmids, contain multiple copies
may be 50 per cell
 A suitable plasmid needs to be present in larger number in a
cell
TYPES OF CLONING VECTOR

-Plasmid
-Bacteriophage
-Cosmid
-Bacterial Artificial Chromosome (BAC)
-Yeast Artificial Chromosome (BAC)
-Human Artificial Chromosome (HAC)
-Retroviral Vectors
PLASMIDS
• Plasmid is an autonomously replicating circular double stranded DNA which can replicate independently
• Plasmids only encode those proteins which are essential for their own replication. These protein-encoding genes are located near
the ori
• Most commonly found in bacteria, sometimes they are present in archaea and eukaryotic organisms
• Size from 1 to over 200 kb.
• Can clone DNA insert of up to 10 kb in size.
• Have high copy number and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent
manipulation
• Low copy number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is
toxic to the cells
Why Plasmids are Good Cloning Vectors:
 Small size (easy to manipulate and isolate)
 Circular (more stable)
 Replication independent of host cell
 Several copies may be present (facilitates replication)
 Frequently have antibiotic resistance (detection easy)
Disadvantages Using Plasmids:
 Cannot accept large fragments(Sizes range from 0 — 10kb)
 Standard methods of transformation are inefficient
NOMENCLATURE OF PLASMID

  pBR322 cloning vector 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 lab
 BACTERIOPHAGES
Consists of DNA (or RNA) carrying number of genes including genes for replication of
phage surrounded by protective coat or capsid made up of proteins Examples: Phage
Lambda, M13 Phage
THE PHAGE INFECTION CYCLE
1. The phage particle attaches to the outside of the bacterium and injects its DNA
chromosome into the cell
2. The phage DNA molecule is replicated, usually by specific phage enzymes coded by
genes in the phage chromosome
3. Other phage genes direct synthesis of the protein components of the capsid, and
new phage particles are assembled and released from the bacterium.
LYTIC CYCLE:
 With some phage types the entire infection cycle is completed very quickly, possibly
in less than 20 minutes.
 This type of rapid infection is called a lytic cycle, as release of the new phage
particles is associated with lysis of the bacterial cell.
 Phage DNA replication is immediately followed by synthesis of capsid proteins, and
the phage DNA molecule is never maintained in a stable condition in the host cell
LYSOGENIC PHASE
 lysogenic infection is characterized by retention of the phage DNA molecule in the host
bacterium, possibly for many thousands of cell divisions
 With many lysogenic phages the phage DNA is inserted into the bacterial genome
 The integrated form of the phage DNA (called the prophage) is quiescent, and a bacterium
(referred to as a lysogen) that carries a prophage is usually physiologically indistinguishable from
an uninfected cell.
 Prophage is eventually released from the host genome and the phage reverts to the lytic mode
and lyses the cell.
 A limited number of lysogenic phages follow a rather different infection cycle.
 GENE ORGANIZATION IN THE λDNA MOLECULE
 The λ DNA molecule is 49 kb in size --- Two forms : linear and circular
 The linear form of phage DNA, with two free ends, and represents the DNA present in the phage
head structure.
COS SITES:
 At either end of the molecule is a short 12-nucleotide stretch in which the DNA is single-
stranded
 The lambda cohesive ends are called the cos sites and they play two distinct roles during the
lambda infection cycle.
 First, they allow the linear DNA molecule that is injected into the cell to be circularized, which is
a necessary prerequisite for insertion into the bacterial genome
 At this stage a large number of new lambda DNA molecules are produced by the rolling circle
mechanism of replication in which a continuous DNA strand is “rolled off” the template
molecule. The result is a catenane consisting of a series of linear lambda genomes joined
together at the cos sites.
 The role of the cos sites is now to act as recognition sequences for an endonuclease that cleaves
the catenane at the cos sites, producing individual lambda genomes.
 This endonuclease, creates the single stranded sticky ends, and also acts in conjunction with
other proteins to package each lambda genome into a phage head structure.

There are two forms in

which lambda DNA exists
1. Linear form
2. Circular form
What is rolling circular mechanism of replication?
Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly
synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of
bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses also replicate
their DNA or RNA via the rolling circle mechanism
MODIFICATION OF λ PHAGES FOR CLONING PURPOSE

Modification of λ phages
 To get a high yield of extracellular λ, the culture must be induced, so that all the bacteria enter
the lytic phase of the infection cycle, resulting in cell death and release of λ particles into the
medium.
 Induction is normally very difficult to control, but most laboratory strains of λ carry a
temperature-sensitive (ts) mutation in the cI gene.
 cl genes are responsible for maintaining the phage in the integrated state (lysogeny)
 If inactivated by a mutation, the cI gene no longer functions correctly and the switch to lysis
occurs
 Although most λ strains are lysogenic, many cloning vectors derived from λ are modified, by
deletions of the cI and other genes, so that lysogeny never occurs.
 These phages cannot integrate into the bacterial genome and can infect cells only by a lytic
cycle
Problems while using λ as cloning vectors
Two problems had to be solved before λ based cloning vectors could be
developed:
PROBLEM #1 : INSERT SIZE
 The total size of lambda genome is 49 kb and it has the capacity to
insert only 2-3kb foreign DNA
 If the total size of the molecule is more than 52 kb, then it cannot be
packaged into the λ head structure and infective phage particles are
not formed.
 This severely limits the size of a DNA fragment that can be inserted into
λ vector
PROBLEM #2 : MULTIPLE RESTRICTION SITES IN GENOME:
 The λ genome is so large that it has more than one recognition
sequence for virtually every restriction endonuclease.
 Restriction cannot be used to cleave the normal λ molecule in a way
that will allow insertion of new DNA, because the molecule would be
cut into several small fragments that would be very unlikely to re-form
a viable λ genome on re-ligation
Solution to problems

Problem #1 (Size ) Solution :

Segments of the λ genome can be deleted without impairing viability
 The λ large segment in the central region of the λ DNA molecule can be removed without
affecting the ability of the phage to infect E. coli cells.
 Removal of all or part of this non-essential region, the size of the resulting lambda
molecule by up to 15-18 kb.
 This means that as much as 18 kb of new DNA can now be added
 This “non-essential” region in fact contains most of the genes involved in integration and
excision of the λ prophage from the E. coli chromosome and lysogeny which we don’t need
 A deleted λ genome is therefore non-lysogenic and can follow only the lytic infection cycle.
 This in itself is desirable for a cloning vector as it means induction is not needed before
plaques are formed
Solution to problems

Problem #2 (Multiple Restriction sites)

Natural selection can be used to isolate modified λ that lack certain restriction sites
 Natural selection used to provide strains of λ that lack the unwanted restriction sites.
 Natural selection can be brought into play by using as a host an E. coli strain that produces
EcoRI.
 Most λ DNA molecules that invade the cell are destroyed by this restriction endonuclease,
but a few survive and produce plaques.
 These are mutant phages, from which one or more EcoRI sites have been lost
spontaneously
 Several cycles of infection will eventually result in λ molecules that lack all or most of the
EcoRI sites.
Overview of constructing our own modified vector
TWO TYPES OF λ VECTORS

There are two types of λ vectors

• Insertion λ vectors
• Replacement λ vectors
INSERTION λ VECTORS

 In insertion vector foreign DNA is inserted without removal of any host lambda DNA
 An insertion vector possesses at least one unique restriction site into which new DNA can
be inserted after an endonuclease generates a cut to open strands
 For instance, EcoRI cuts at cl gene ( G|AATTC)

For example:
1. λ gt10
2. λ ZAPII
REPLACEMENT λ VECTORS

 In replacement vector foreign DNA is inserted and removal of any unwanted host lamda
DNA also occurs
 A λ replacement vector has two recognition sites for the restriction endonuclease
 The unwanted DNA removed from these sites is called stuffer fragment
 These sites flank a segment of DNA that is replaced by the DNA to be cloned
 Replacement vectors are generally designed to carry larger pieces of DNA than insertion
vectors can handle.

For example:
λ EMBL4
The end