TOPIC OF TERM PAPER

WHY COSMIDS ARE

USED AS VECTOR?

SUBMITTED TO: SUBMITTED BY:

MR. HARSH VISHAKHAWADHAWAN

(LECTURER IN (CLASS-
BIOTECHNOLOGY) B-TECH BIO TECH)
ROLL NO.-R108A06
(REGISTRATION NO.
1040070130)
 ACKNOWLEDGEMENT

I feel immense pleasure to give the credit of my term paper work not only to one individual
as this work is integrated effort of all those who concerned with it. I want to owe my thanks
to all those individuals who guided me to move on the track.

I sincerely expressed my gratitude and lot of thanks to Mr. Harsh Lecturer of

Concepts of Biotechnology, Lovely Professional University for helping me in completing my
term paper work and making it a great success.

Last but not least i would thank all my friends and faculty members who
rendered their precious time for contributing their skills which made my term paper more
appealing and attractive.
 CONTENTS
• INTRODUCTION

• BODY

CLONING VECTORS

PLASMID VECTOR

BACTERIOPHAGE VECTORS

COSMID VECTOR

• COSMIDS ARE MORE EFFECIENT THAN PLASMID AND PHAGE

VECTOR

LARGE DNA FRAGMENTS CAN BE CLONED IN COSMIDS

• SUMMARY

• BIBLIOGRAPHY
 INTRODUCTION

A recombinant DNA molecule is a vector(eg a plasmid, phage or virus, cosmid) into which
the desired DNA fragment has been inserted to enable its cloning in an appropriate host to
achieve the recombinant DNA, the DNA segment are integrated into an autonomously
replicating DNA molecule called vector; most commonly used vectors are plasmids, phage or
virus, cosmid. The vectors containing DNA segments to be cloned called DNA inserts are
then introduced into a suitable organism, usually a bacterium; this organism is called host
while the process is called transformation. Vector consist of polylinker that recognise several
different restriction enzymes, drug resistant gene, and a replication origin. Cosmids are
essentially plasmids that contain a minimum of 250bp of DNA and COS site from lambda
phage. Cosmids has

(1) High transformation efficiency

(2) The cosmid can carry up to 45kb whereas plasmid and phage vectors are limited to 25kb.
 VECTOR

"Vector" is an agent that can carry a DNA fragment into a host cell. If it is used for reproducing the DNA
fragment, it is called a "cloning vector". If it is used for expressing certain gene in the DNA fragment, it is
called an expression vector.

Commonly used vectors include plasmid, lambda vector, cosmid etc.

PLASMID
Plasmids are circular, double-stranded DNA molecules that exist in bacteria and in the nuclei of some
eukaryotic cells. They can replicate independently of the host cell. The size of plasmids ranges from a few
kb to near 100 kb.

It contains a polylinker which can recognize several different restriction enzymes, an ampicillin-resistance
gene (ampr) for selective amplification, and a replication origin (ORI) for proliferation in the host cell.

A plasmid vector is made from natural plasmids by removing unnecessary segments and adding essential
sequences. To clone a DNA sample, the same restriction enzymes must be used to cut both the vector and
the DNA sample. Therefore, a vector usually contains a sequence (polylinker) which can recognize several
restriction enzymes so that the vector can be used for cloning a variety of DNA samples.

A plasmid vector must also contain a drug-resistance gene for selective amplification. After the vector
enters into a host cell, it may proliferate with the host cell. However, since the transformation efficiency of
plasmids in E. coli is very low, most E. coli cells that proliferate in the medium would not contain the
plasmids. Therefore, we must find a way to allow only the transformed E. coli to proliferate. Typically,
antibiotics are used to kill E. coli cells which do not contain the vectors. The transformed E. coli cells are
protected by the ampicillin-resistance gene (ampr) which can express the enzyme, β -lactamase, to
inactivate the antibiotic ampicillin.
 λ − Phages
λ Phages are viruses that can infect bacteria. The major advantage of the λ phage vector is its high
transformation efficiency, about 1000 times more efficient than the plasmid vector.

Schematic drawing of the DNA cloning using λ phages as vectors. The DNA to be cloned is first inserted
into the λ DNA, replacing a nonessential region. Then, by an in vitro assembly system the λ virion
carrying the recombinant DNA can be formed. The λ genome is 49 kb in length which can carry up to 25
kb foreign DNA.

The assembly process of the λ virion.

The extreme ends of the λ DNA are known as COS sites, each are single stranded, 12 nucleotides long.
Because their sequences are complementary to each other, one end of λ DNA may base-pair with the other
end of a different λ DNA, forming concatemers. The two ends of a λ DNA may also bind together,
forming a circular DNA. In the host cell, the λ DNA circularizes because ligase may seal the join of the
COS sites.

In the assembly process of λ virions, two proteins Nu1 and A can recognize the COS site, directing the
insertion of the λ DNA between them into an empty head. The filled head is then attached to the tail,
forming a complete λ virion. The whole process normally takes place in the host cell. However, to
prepare the λ virion carrying recombinant λ DNA, the following in vitro assembly system is commonly
used.

Proteins Nu1 and A are encoded by the genes in the λ genome. If the two genes are mutated, λ DNA
cannot be packaged into the pre-assembled head. Because tails attach only to filled heads, the cell will
accumulate separate empty heads and tails, which can then be extracted. When the extract is mixed with
recombinant λ DNA and proteins Nu1 and A, the complete λ virion carrying recombinant λ DNA will
be assembled.

COSMIDS
Cosmids are essentially plasmids that contain a minimum of 250 bp of lambda DNA, which includes some
sequences from phage genome: (1) the cos site (2)sequences needed for binding of and cleavage by
terminase so that under appropriate conditions they are packaged in vitro into empty lambda phage particles.

• High transformation efficiency.

• The cosmid vector can carry up to 45 kb whereas plasmid and λ phage vectors are limited to 25 kb.

Cloning by using cosmid vectors. (a) In addition to ampr, ORI, and polylinker as in the plasmid vector, the
cosmid vector also contains a COS site. (b) After cosmid vectors are cleaved with restriction enzyme, they
are ligated with DNA fragments. The subsequent assembly and transformation steps are the same as
cloning with λ phages.
 COSMIDS ARE MORE EFFICIENT THAN PLASMIDS AND

BACTERIPHAGES
Most DNA cloning is done with E. coli plasmid vectors because of the relative simplicity of the cloning
procedure. However, the number of individual clones that can be obtained by plasmid cloning is limited by the
relatively low efficiency of E. coli transformation and the small number (only a few hundred) of individual
transformed colonies that can be grown on a typical culture plate. These limitations make plasmid cloning of
all the genomic DNA of higher organisms impractical. For example, ≈1.5 × 105 clones carrying 20-kb DNA
fragments are required to represent the total human haploid genome, which contains ≈3 × 109 base pairs.
Fortunately, cloning vectors derived from various bacteriophages have proved to be a practical means for
obtaining the required number of clones to represent large genomes. A collection of clones that includes all the
DNA sequences of a given species is called a genomic DNA library, or simply genomic library. Once a
genomic library is prepared, it can be screened for clones containing a sequence of interest.

Bacteriophage λ Can Be Modified for Use as a Cloning Vector and Assembled in

Vitro

Bacteriophage λ is probably the most extensively studied bacterial virus, and a great deal is known
about its molecular biology and genetics. A λ phage virion has a head, which contains the viral
DNA genome, and a tail, which functions in infecting E. coli host cell. When λ DNA enters the
host-cell cytoplasm following infection, it undergoes either lytic or lysogenic growth. In lytic
growth, the viral DNA is replicated and assembled into more than 100 progeny virions in each
infected cell, killing the cell in the process and releasing the replicated virions. In lysogenic growth,
the viral DNA inserts into the bacterial chromosome, where it is passively replicated along with the
host-cell chromosome as the cell grows and divides.

The λ genes encoding the head and tail proteins as well as various proteins involved in the lytic and
lysogenic growth pathways are clustered in discrete regions of the ≈50-kb viral genome. When
bacteriophage λ is used as a cloning vector, it must be capable of lytic growth, but other viral
functions are irrelevant. Consequently, the genes involved in the lysogenic pathway and other viral
genes not essential for the lytic pathway are removed from the viral DNA and replaced with the
DNA to be cloned. Up to ≈25 kb of foreign DNA can be inserted into the λ genome, resulting in a
recombinant DNA that can be packaged in vitro to form virions capable of replicating and forming
plaques on E. coli host cells.

During the in vivo assembly of λ virions within infected host cells, viral heads and tails initially are
assembled separately, from multiple copies of the various proteins that compose these complex
structures. Replication of λ DNA in a host cell generates long multimeric DNA molecules, called
concatomers, that consist of multiple copies of the viral genome linked end to end and separated by
specific nucleotide sequences called COS sites. Two λ proteins, designated Nu1 and A, bind to COS
sites and direct insertion of the DNA lying between two adjacent COS sites into a preassembled
head. This process results in the packaging of a single ≈50-kb λ genome from the multimeric
concatomer into each preassembled head. Host-cell chromosomal DNA is not inserted into the λ
heads because it does not contain any copies of the COS sequence. Once λ DNA is inserted into a
preassembled λ head, the preassembled tail is attached, producing a complete virion.

To prepare infectious λ virions carrying recombinant DNA, the phage-assembly process is carried
out in vitro. In one method, E. coli cells are infected with a λ mutant defective in A protein, one of
the two proteins required for packaging λ DNA into preassembled phage heads. These cells
accumulate preassembled “empty” heads; since tails attach only to heads “filled” with DNA.
Preassembled tails also accumulate in these cells. An extract containing high concentrations of
empty heads and tails is prepared by lysing cells infected with the λ A mutant. When this extract is
mixed with isolated A protein (obtained from λ-infected cells) and recombinant λ DNA containing a
COS site, the DNA is packaged into the empty heads. The tails in the extract then combine with the
filled heads, yielding complete virions carrying the recombinant λ DNA.

This procedure produces a high yield of recombinant virions that are fully infectious and can
efficiently infect E. coli cells. Each virion particle binds to receptors on the surface of a host cell
and injects its packaged recombinant DNA into the cell. Infection by λ phage is about a thousand
times more efficient than transformation with plasmid vectors, accounting for the high
efficiency of λ phage cloning. For instance, ≈106 transformed colonies per microgram of
recombinant plasmid DNA can be obtained routinely, whereas ≈109 plaques representing λ
clones can be obtained per microgram of recombinant λ DNA.

Nearly Complete Genomic Libraries of Higher Organisms Can Be Prepared by λ Cloning

With the availability of λ phage cloning vectors, preparation of genomic libraries for higher
organisms, including humans, is feasible. A genomic library is a set of λ (or plasmid) clones that
collectively contain every DNA sequence in the genome of a particular organism. The λ DNA first
is treated with a restriction enzyme to produce fragments called λ vector arms, which have sticky
ends and together contain all the genes necessary for lytic growth. This step frees the nonessential
region in the middle of the λ genome; this region is separated from the λ arms and discarded.
Genomic DNA then is extracted from a cell type that contains all the genetic information of the
organism under study. Sperm cells or cells of an early embryo often are used as sources of
mammalian DNA. The extracted DNA then is cleaved by a restriction enzyme to produce ≈20-kb
fragments with sticky ends complementary to the sticky ends on the λ vector arms being used.

The λ arms and the collection of genomic DNA fragments are mixed in about equal amounts. The
complementary sticky ends on the fragments and λ arms hybridize and then are joined covalently by
DNA ligase. Each of the resulting recombinant DNA molecules contains a foreign DNA fragment
located between the two arms of the λ vector DNA. The ligated recombinant DNAs then are
packaged into λ virions in vitro as described above. Only DNA molecules of the correct size can be
packaged to produce fully infectious recombinant λ virions.

Finally, the recombinant λ virions are plated on a lawn of E. coli cells to generate a large number of
recombinant λ plaques. Since each plaque arises from a single recombinant virion, all the progeny λ
phages that develop are genetically identical and constitute a clone carrying a particular genomic
DNA insert. The different plaques correspond to distinct phage clones, each carrying a different
DNA insert, and collectively they constitute a λ genomic library. Multiple λ vectors have been
constructed containing different restriction sites, so that restriction fragments generated by a variety
of restriction enzymes can be cloned in λ vectors.

In constructing a library of genomic DNA, the DNA commonly is cleaved with a restriction enzyme
that recognizes a 4-bp restriction site. A specific 4-bp sequence will occur on average once every 44
= 256 base pairs. Complete digestion of the human haploid genome, which contains ≈3 × 109 base
pairs, would yield somewhat more than 107 nonoverlapping different fragments. However, to
increase the probability that all regions of the genome are successfully cloned and represented in the
λ genomic library, the genomic DNA usually is only partially digested to yield overlapping
restriction fragments of ≈20 kb. In a large λ library constructed from such overlapping restriction
fragments, a specific sequence of genomic DNA (e.g., the gene encoding β-globin) often is
contained in or extends over several “overlapping” clones. By a technique called chromosome
walking, described in the next chapter, overlapping genomic clones can be ordered.

The size of a genomic library for a given organism depends on the amount of DNA in that
organism's haploid genome. If the human genome of about 3 × 10 9 base pairs is cleaved into 20-kb
fragments for insertion into a λ vector, then roughly 1.5 × 105 different recombinant λ phage virions
would be required to constitute a complete library. Because the restriction fragments of human
DNA are incorporated into phages randomly, about 106 recombinant phages are necessary to assure
that each region of human DNA has a 90 – 95 percent chance of being included.

Each plaque produced by a recombinant bacteriophage λ contains large numbers of recombinant

virions and, consequently, large numbers of identical cloned DNA fragments. Hybridization
methods for identifying recombinant λ clones of interest are described in a later section. Because
these methods allow specific detection of very small plaques, as many as 5 × 10 4 plaques can be
screened on a single culture plate. Thus only 20 – 30 petri dishes, each containing about 5 × 104 λ
plaques, are sufficient to represent the entire human genome. In contrast, to screen 106 recombinant
plasmids carrying the entire human genome would require about 5000 petri dishes, because only
200 or so transformed E. coli colonies can be detected on a typical Petri dish.
Larger DNA Fragments Can Be Cloned in Cosmids

Both λ phage vectors and the more commonly used E. coli plasmid vectors are useful for cloning
DNA fragments up to ≈20 – 25 kb. However, cloning of much larger fragments is desirable for
sequencing of extremely long DNAs such as the DNA in a eukaryotic chromosome. Also, because
of the common occurrence of large introns in genes from higher eukaryotes, it is often necessary to
clone DNA fragments greater than 25 kb in order to include an entire gene in one clone.
Consequently, additional types of cloning vectors have been developed for cloning larger fragments
of DNA.

One common method for cloning larger fragments makes use of elements of both plasmid and λ
phage cloning. In this method, called cosmid cloning, recombinant plasmids containing inserted
fragments up to 45 kb long can be efficiently introduced into E. coli cells. A cosmid vector is
produced by inserting the COS sequence from λ phage DNA into a small E. coli plasmid vector
about 5 kb long. Like other plasmid vectors discussed earlier, cosmid vectors contain a replication
origin (ORI), an antibiotic-resistance gene (e.g., ampr), and a polylinker sequence containing
numerous restriction-enzyme recognition site. Next, the cosmid vector is cut with a restriction
enzyme and then ligated to 35- to 45-kb restriction fragments of foreign DNA with complementary
sticky ends. If the concentration of foreign DNA is high enough, the ligation reaction generates long
DNA molecules containing multiple restriction fragments of the foreign DNA separated by the 5-kb
cosmid DNA. These ligated DNA molecules, which resemble the concatomers that form during
replication of λ phage in a host cell, can be packaged in vitro as described earlier.

In the packaging reaction, the λ Nu1 and A proteins bind to COS sites in the ligated DNA and direct
insertion of the DNA between two adjacent COS sites into empty phage heads. Packaging will
occur so long as the distance between adjacent COS sites does not exceed about 50 kb (the
approximate size of the λ genome). Phage tails then are attached to the filled heads, producing viral
particles that contain a recombinant cosmid DNA molecule rather than the λ genome. When these
virions are plated on a lawn of E. coli cells, they bind to phage receptors on the cell surface and
inject the packaged DNA into the cells.

Since the injected DNA does not encode any λ proteins, no viral particles form in infected cells and
no plaques develop on the plate. Rather, the injected DNA forms a large circular plasmid, composed
of the cosmid vector and an inserted DNA fragment, in each host cell. This plasmid replicates and is
segregated to daughter cells like other E. coli plasmids, and the colonies that arise from transformed
cells can be selected on antibiotic plates. The high efficiency of λ phage infection of E. coli cells
makes cosmid cloning a practical method of generating plasmid clones carrying DNA fragments up
to 45 kb long. Since many genes of higher eukaryotes are on the order of 30 – 40 kb in length,
cosmid cloning increases the chances of obtaining DNA clones containing the entire sequences of
genes.

Another strategy similar to cosmid cloning makes use of larger E. coli viruses such as bacteriophage
P1, whose head can accommodate larger DNA molecules than the λ phage head. Recombinant
plasmids containing DNA fragments up to ≈100 kb long can be packaged in vitro with the P1
system.

SUMMARY

• λ phage cloning is more complex than plasmid cloning, but it has the advantage of producing far
more clones per microgram of DNA. Also far more λ plaques than E. coli colonies can be plated
and detected on a single culture plate, simplifying the storage and screening of large numbers of
clones.

• The middle region of λ DNA contains genes that are not required for lytic replication. In λ vector
DNA this region is flanked by restriction sites. To produce recombinant virions, λ vector DNA is
digested with an appropriate restriction enzyme, removing the nonessential middle region and
generating the right and left vector arms with sticky ends. DNA fragments up to ≈25 kb in length
then are ligated to the λ arms. Finally, recombinant λ DNA is packaged into λ virions in vitro

• Recombinant λ virions assembled in vitro are used to infect a lawn of E. coli cells, producing one
plaque for each recombinant λ phage. Each plaque contains large numbers of recombinant λ virions,
all descended from the single recombinant λ phage that initiated the plaque. The high efficiency of λ
cloning results from the ability to package recombinant λ DNA in vitro and the high infectivity of
the resulting virions.

• A genomic library is a set of λ or other clones prepared from the total genomic DNA of an
organism. A genomic library must contain a sufficiently large number of independently derived
clones that the probability is high (>95%) that every DNA sequence of the organism is represented
in the library. A library of the human genome composed of λ clones contains about 106 recombinant
phages, which can be concentrated into a single microliter of buffer.

• DNA fragments up to ≈25 kb in length can be cloned in plasmid and λ vectors. Other vectors must
be used for cloning larger DNA fragments. These include cosmid and P1 vectors, bacterial artificial
chromosomes (BACs), and yeast artificial chromosomes (YACs).
BIBLIOGRAPHY
• Google.com

• Blackle.com

• msn.com

• Biotechnology Expanding Horizons-

Author - B.D. Singh

Kalyani Publishers