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Lambda phage vector

BIOPSYCHOLOGY (University of Madras)

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BACTERIOPHAGE DERIVED VECTORS

COMMON INTRO FOR ALL PHAGE VECTORS

Bacteriophages, or phages as they are commonly known, are viruses that specifically infect
bacteria. Like all viruses, phages are very simple in structure, consisting merely of a DNA (or
occasionally ribonucleic acid (RNA)) molecule carrying a number of genes, including several for
replication of the phage, surrounded by protective coat or capsid made up of protein molecules.
Fred Blatter and his colleagues were the first to develop a bacteriophage as vector.

The general pattern of infection, which is the same for all types of phage, is a three-step
process:
1. The phage particle attaches to the outside of bacterium and injects its DNA chromosome into
the cell.
2. The phage DNA molecule is replicated, usually by specific phage enzymes coded by genes on
the phage chromosome.
3. Other phage genes direct synthesis of protein components of capsid, and new phage particles
are assembled and released from the bacterium.
With some phage types the entire infection cycle is completed very quickly, possibly in less than
20min. This type of rapid infection is called a lytic cycle, as release of the new ph age particles is
associated with lysis of the bacterial cell.
The characteristic feature of a lytic infection cycle is that 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. In contrast to a lytic cycle, lysogenic infection is characterized
by retention of the phage DNA molecule in the host bacterium, possibly for many thousands of
cell divisions.

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PHAGE VECTORS
Bacteriophage lambda vectors were
developed because several observations
were made that suggested that they could
complete their life cycles even if foreign
DNA was inserted into a portion of its
genome. This suggested that certain
regions of the virus were not essential.

The life cycle of lambda.

1. 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
2. Penetration - DNA is injected into the cell; at this point it can enter one of two
pathways;
o 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
o Lytic pathway - large
scale production of
bacteriophage particles
that eventually leads to
the lysis of the cell; base
pairing at the cos site
leads toa circular
molecule
3. Early transcription -
transcription proceeds from
the pL and pR promoters,
through the N and crogenes and
stops at terminators tL and tR1;
a low level of transcription
through the O and P genes
occurs and terminates at tR2;

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the N product is an antitermination factor that is important for the next stage of
transcription
4. Delayed early transcription - the N product binds to RNA polymerase and transcription
proceeds past the tL, tR1 and tR2 terminators; genes to the left of N, involved in
recombination, to the right of cro, involved in replication, are expressed at this point;
another protein expressed from the Q gene is used for antitermination of later
transcription
5. Replication - early replication is through a theta form initiated from a single origin of
replication site; later replication is via rolling circle replication; this produces long
concatamers of the phage DNA that are cleaved at the cosL and cosR sites
6. Late transcription - the protein product of the crogene builds up to a critical level and
then binds to the oL and oR to stop early transcription; another protein, a product of the
Q gene, has built up and activates transcription at the p'R promoter by antitermination;
transcription terminates with in the bregion; this transcription results in the production
of the proteins required for the head and tail of the mature phage particle and those
required for bacterial cell lysis
7. Assembly - a prophage head is produced; a unit length DNA is placed into the head by
the action of the Nu1 and A proteins; the DNA is locked into place by the D protein
and ter function of the A protein clips the DNA at the cosL and cosR sites; the
concatamer is released, the tail is added and the mature phage particle is completed
Packaging of the DNA into the 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.

Two important developments suggested that lambda may be suitable as a cloning vector. First it
was determined that the gene products between the J and N genes could be removed and the
life cycle could be completed. Second, restriction enzyme sites could be eliminated which
permitted the development of a vector with a single site for insertion of foreign DNA. Two types
of vectors have been developed:

GENOME OF LAMBDA PHAGE

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The lambda DNA molecule is 49 kb in size.

It is a temperate phase and this can carry out lytic and lysogenic cycles. The positions and
identities of most of the genes on the lambda DNA molecule are known (Fig. 4.28).

Lambda phage can have both linear and circular forms of DNA. The molecule shown in (Fig.
4.28) is linear, with two free ends, and represents the DNA present in the phage head. This linear
molecule consists of two complementary strands of DNA, base paired according to the Watson-
Crick rules.’ However, at either end of the molecule is a short 12-nucleotide stretch in which the
DNA is single-stranded [Fig. 4.30(a)].
The two single strands are complementary, and so can base pair with one another to form a
circular, completely double-stranded molecule [Fig. 4.30(b)]. Complementary single strands are
often referred to as ‘sticky’ ends or cohesive ends, because base pairing between them can ‘stick’
together the two ends of a DNA molecule (or the ends of two different DNA molecules). The
lambda cohesive ends are called the cos sites and they play two distinct roles during the lambda
infection cycle

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ROLE OF COS SITE

 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
 The second role of the cos sites is rather different, and comes into play after the pro-ph-
age has excised from the host 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 A genomes joined together at the cos sites.
 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,
which is the product of gene A on the lambda DNA molecule, creates the single stranded
sticky ends, and also acts in association with other proteins to package each lambda
genome into a phage head structure.

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TWO PROBLEMS HAVE TO BE SOLVED BEFORE LAMBDA- BASED

CLONING VECTORS COULD BE DEVELOPED:
 1. The lambda molecule can be increased in size by only about 5%, representing the ad-
dition of only 3kb of new DNA. If the total size of the molecule is more than 52kb, then
it cannot be packaged into the lambda head structure and infective phage particles are not
formed. This severely limits the size of a DNA fragment that can be inserted into an
unmodified lambda vector.
 2. The lambda genome is so large that it has more than one recognition sequence for
virtually every restriction endonucleases. Restriction cannot be used to cleave the normal
lambda molecule in a way that will allow insertion of new DNA, because the molecules
would be cut into several small fragments that would be very unlikely to reform a viable
lambda genome on relegation.

Due to these reasons the DNA of naturally occurring lambda phage cannot be used as a
cloning vector.

Solving problem 1:
From research it has been found out that large segment in the central region of the lambda DNA
molecule can be removed without affecting the ability of the phage to infect E. coli cells.
Removal of this nonessential region between positions 20 and 35 on the map decreases the size
of the lambda genome by up to 15kb.
This makes a room for as much as 18kb of new DNA which can be added to it to form a
recombinant molecule.
This non-essential genes thus removed are involved in integration and excision of the lambda
pro-phage from the E. coli chromosome.
Solving problem 2: We can remove unnecessary restriction sites by carrying out in vitro
mutagenesis. For example, an ECoRI site, GAATTC, could be changed to GGATTC, which is
not recognized by the enzyme.
TYPES OF LAMBDA VECTORS:
There are two types of lambda cloning vectors.

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(a) Lambda Insertion Vectors:

 In this case 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. The size of the DNA fragment that an individual
vector can carry depends on the extent to which the non-essential region has been
deleted, e.g.; lambda-gtl0, lambda- ZAP11.


(b) Lambda Replacement Vectors:

 These vectors have two recognition sites for the restriction endonucleases. These sites
flank a segment of DNA that is replaced by the DNA to be cloned [Fig.4.34(a)]. Often
the replaceable fragment (or stuffer fragment) carries additional restriction sites that can
be used to cut it up into small pieces so that its own reinsertion during a cloning
experiment is very unlikely.
 Replacement vectors are generally designed to carry large pieces of DNA than insertion
vectors can handle e.g., lambda- EMBL, lambda-GEMll, etc.

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CLONING EXPERIMENTS WITH LAMBDA INSERTION OR REPLACEMENT

VECTORS:

A cloning experiment with a lambda vector can be carried out by following the similar method
that we followed for a plasmid vector—the lambda DNA molecules are digested with suitable
restriction endonuclease enzyme, the gene of interest is added, the mixture is ligated and the
resulting recombinant DNA is introduced into E. coli host cell [Fig. 4.35(a)].
This type of experiment requires that the vector be in its circular form, with the cos sites
hydrogen bonded to each other.
The process which requires a circular lambda DNA molecule is not particularly efficient in
transformation hence . a linear form of the vector is more preferred for high transformat
efficiency. When the linear form of the vector is digested with the relevant restriction
endonuclease, the left and right arms are released as separate fragments.
A recombinant DNA can be constructed by mixing together our gene of interest with the vector
arms [Fig. 4.35(b)]. Ligation results in several molecular arrangements, including catenae’s
comprising left arm-DNA-right arm repeated many times. Recombinant phage thus produced in
the test tube can be used to infect an E.coli culture.

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VISUALIZATION OF PHAGE INFECTION AFTER THE PROCESS OF

TRANSFECTION:
The entry of recombinant DNA in the host cell is followed by the lytic cycle which eventually
results in the lysis of the host cell. The lysed host cell can be located on the agar medium as
plaques on a lawn of bacteria. Each plaque is a zone of clearing produced as the phages lyse the
cell and move on to infect the neighbouring bacteria.

SCREENING OF TRANSFORMED HOST CELLS USING BACTERIOPHAGE

LAMBDA VECTORS:
A variety of ways could be employed to distinguish between recombinant plaques from non-
recombinant ones.
(a) Insertional Inactivation of Lac Z’ Gene Carried by the Lambda Phage Vector:
Insertion of our gene of interest into the lac Z’ gene inactivates beta- galactosidase synthesis.
Recombinants are distinguished by plating cells on X-gal agar where the recombinant plaques

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are clear whereas non-recombinant plaques are blue in colour.

(b) Insertional Inactivation of Lambda Cl Gene:
Several lambda cloning vectors have restriction site in the cl gene. Insertional inactivation of cl
gene cause a visible change in the plaque morphology. Normal plaques appear turbid (hazy)
whereas recombinant plaques with disrupted cl gene are clear.
(c) Selection using Spi Phenotype:
P2 phage is a relative of lambda phage, lamda phages cannot infect E. coli cells that already has
an integrated P2 phage in its genome. Due to this, lamda phage is said to be Spi-+ (sensitive to
P2 pro-phage infection). Some lambda cloning vectors are designed so that insertion of new
DNA causes a change from Spi-+ to Spi-–, enabling the recombinant to infect cells that carry P2
pro-phages
Uses of Bacteriophage Lambda Vectors:
The main use of all lambda based vectors is to clone DNA fragments that are too long to be
handled by plasmid or M13 vectors. Lambda-EMBL4 can carry up to 20kb of our gene of
interest.
Advantages of Bacteriophage Lambda Vectors
1. Storage of phage particles is comparatively much easier than that of plasmid based vectors.
2. The shelf-life of phage particles is infinite.
3. Transformation of E. coli is much easier with phage particles.
Disadvantages of Bacteriophage Lambda Vectors:
 There is still no truly rapid, reliable protocol for the production of very clean lambda-
DNA.
 Even in the replacement vectors, almost two thirds of the DNA is made up of vector
sequences. If possible, you should clone the sections that are of interest using plasmids.

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