Cosmid vectors 

are hybrids between plasmid and phage λ vectors. The classic example
of cosmid vector is c2RB, which carries an origin of replication and a cloning site and has antibiotic-
resistant genes. As with the phage λ vector, the cosmid vector encodes the cos sequences required for
packaging of DNA into λ capsid. Cosmid vectors are designed to clone large fragments of DNA and
to grow their DNA as a virus or as a plasmid. Cosmid vectors are used in homologous
recombination between two different plasmids in the same cell and grown in both bacteria and animal
cells. Cosmid vectors, along with λ vectors, are used as standard vectors for the cloning of genomic
DNA

As we have become more sophisticated in our ability to manipulate and characterize DNA
sequences, new vector systems have been developed for specific uses. This is a short look at some of
these other systems.

A major thrust of vector development has been to create vectors that will handle larger foreign DNA
inserts, aiming to reduce the number of recombinants it is necessary to look at in order to identify a
specific DNA sequence.

Cosmid vectors were among the first large insert cloning vehicles developed.

 
A cosmid is a plasmid that contains a cos site
from the lambda genome.

The vector replicates as a plasmid

(it contains a ColE1 origin of replication),
uses Ampr for positive selection and
employs lambda phage packaging to select for
recombinant plasmids carrying foreign DNA
inserts 45 KB in size.

Ligation of cosmid vector and foreign DNA

fragments (SauIIIA partial digest fragments
45 kb in size) is similar to ligation into a
lambda substitution vector.
The desired ligation product is a concatemer
of
45 kb foreign DNA fragment and 5 kb cosmid
vector sequences.

This concatemer is then packaged into viral particles (remember packaging is cos site to cos site)
and these are used to infect E. coli where the cosmid vector replicates using the ColE1 orignin of
replication. Phage packaging serves only to select for recombinant molecules and to transfer
 these long DNA molecues (50 kb total) into the bacterial host (50 kb fragments transform very
inefficiently while phage infection is very efficient).

 
Very Large Prokaryotic Vector Systems

Recently, the need for prokaryotic vectors capable of replicating very large foreign DNA
fragments
(> 1 MB in size) has produced a couple of additional prokaryotic vector systems.
One is based on another bacteriophage called P1.
P1 phage can carry in excess of 100 KB of foreign DNA but do not seem to have found wide
acceptance.

A few years ago, a Bacterial Artificial Chromosome (BAC) vector was developed allowing
foreign DNA fragements over 1 MB to be propagated in E. coli.
These BAC vectors replicate as low copy number plasmids (to minimize the demands on the host
replication system) but how do we get such large DNA fragments into E. coli?

Standard methods for transforming E coli rely on the chemical preparation of the E coli to put
them in a 'transformation competent' state.
This typically involves incubating the bacteria in the presence of divalent cations (particularly
Ca+2) at low temperature (4 oC) which puts the cell membrane into a 'semi-crystaline state.
Addition of DNA to these 'competent cells' allow the DNA to bind to the semi-crystaline
membrane. Heat shocking the cells (42 oC) remobilizes the membrane and allows the DNA to
cross the membrane. This transit across the membrane is sensitive to DNA length - small
plasmids cross the membrane rapidly while larger molecules are unable to cross.

Getting very large molecules across the cell membrane relies on an alternative mechanism called
electroporation. This involves mixing DNA with E coli cells and then exposing the mixture to a
high-voltage pulse. The high voltage induces massive membrane rearrangements and the
coincident uptake of DNA which is independant of size. BACs have been an important tool for
the human genome sequencing project.

Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is

limited, as a host (usually E. coli) can only contain one fosmid molecule. Fosmids can hold DNA
inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library
is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid
vector.[1] The ligation mix is then packaged into phage particles and the DNA is transfected into the
bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher
stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful
for constructing stable libraries from complex genomes. Fosmids have high structural stability and
have been found to maintain human DNA effectively even after 100 generations of bacterial growth.
[2]
 Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence. [3]
The fertility plasmid or F-plasmid was discovered by Esther Lederberg and encodes information for
the biosynthesis of sex pilus to aid in bacterial conjugation. Conjugation involves using the sex pilus
to form a bridge between two bacteria cells; the bridge allows the F+ cell to transfer a single-stranded
copy of the plasmid so that both cells contain a copy of the plasmid. On the way into the recipient
cell, the corresponding DNA strand is synthesized by the recipient. The donor cell maintains a
functional copy of the plasmid. It later was discovered that the F factor was the first episome and can
exist as an independent plasmid making it a very stable vector for cloning. Conjugation aids in the
formation of bacterial clone libraries by ensuring all cells contain the desired fosmid. [4]
Fosmids are DNA vectors that use the F-plasmid origin of replication and partitioning mechanisms to
allow cloning of large DNA fragments. A library that provides 20–70-fold redundant coverage of the
genome can easily be prepared.

Uses[edit]
Sometimes it is difficult to accurately distinguish individual chromosomes based on chromosome
length, arm ratio, and C-banding pattern. Fosmids can be used as reliable cytological markers for
individual chromosome identification and fluorescent in situ hybridization based metaphase
chromosome karyotypes can be used to show whether the positions of these fosmids were
successfully constructed.[8]
The fosmid system is excellent for rapidly creating chromosome-specific mini-BAC libraries from
flow-sorted chromosomal DNA. The major advantage of Fosmids over other cosmid systems lies in
its capability of stably propagating human DNA fragments. [9] Highly repetitive in nature, human DNA
is well known for its extreme instability in multicopy vector systems. It has been found that the
stability increases dramatically when the human DNA inserts are present in single copies in
recombination deficient E. coli cells. Therefore, Fosmids serve as reliable substrates for large scale
genomic DNA sequencing.
SHUTTLE VECTOR

 A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different
host species. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two
different cell types. The main advantage of these vectors is they can be manipulated in E. coli and
then used in a system which is more difficult or slower to use (e.g., yeast, other bacteria).Shuttle
vectors include plasmids that can propagate in eukaryotes and prokaryotes (e.g., both Saccharomyces
cerevisiae and Escherichia coli) or in different species of bacteria (e.g., both E. coli and Rhodococcus
erythropolis). There are also adenovirus shuttle vectors, which can propagate in E. coli and mammals.
Shuttle vectors are frequently used to quickly make multiple copies of the gene in E. coli
(amplification). They can also used for in vitro experiments and modifications (e.g., mutagenesis,
PCR).
Eukaryotic Vector Systems

Numerous vector systems have been developed for use in eukaryotic systems.
The most common of these are often called 'shuttle vectors' as they replicate in both prokaryotic and
eukaryotic hosts. In general, DNA manipulations and characterizations are done in prokaryotic
systems, and then the manipulated DNA is reintroduced to eukaryotic systems for functional analysis.
Yeast Vectors
Shuttle vectors were first developed for transfering DNA fragments between E coli and S cerevisiae.
These vectors contain an antibiotic resistance gene for positive selection in E coli, and E coli origin of
replication (ColE1 ori), and a polylinker in the lacZ alpha-complementing fragment for insertional
inactivation by the foreign DNA insert. In addition, the vector contains a yeast specific origin of
replication (derived from the naturally occuring yeast plasmid called the 2 um circle - 2um ori)
and amino-acid or N-base biosynthsis enzymes for positive selection in yeast (HIS, LEU and ADE
genes)

Mammalian Vectors
Shuttle vectors have also been developed for use in mammalian tissue culture.
Like the yeast shuttle vectors, mammalian shuttle vectors require sequeces enabling them to replicate
in mammalian tissue culture. These eukaryotic origins of replication are typically derived from well
characterized mammalian viruses - most commonly SV-40 (SV-40 ori and Large T antigen system) or
Epstein-Barr virus (mononucleosis). Both systems allow the vector to replicate within the host cell as
a plasmid without integrating into the genome. In addition to the origin of replication, these shuttle
vectors also carry antibiotic resistance genes which function in eukaryotic cells (ie neomycin (G418)
resistance, hygromycin resistance, methotrexate resistance etc).

Finally, artificial chromosome vectors have been developed for use in yeast (YACs) and are being
developed for use in mammalian cells (MACs).
These eukaryotic vectors contain telomeric sequences (eukaryotic chromsomes are linear not circular
molecules) and centromeres to ensure appropriate segregation of the artificial chromosomes as well as
selectable markers. Such vector systems may gain in importance as we move towards manipulating
the eukaryotic genome.