Shuttle Vector DNA molecule originating from a virus, a plasmid, or the cell of a higher organism into which another DNA fragment of appropriate size can be integrated without the loss of the vector capacity for self-replication. Vectors introduce foreign DNA into the host cells, where it can be reproduced in large quantities. A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. It include plasmids that can propagate in eukaryotes and prokaryotes or in different species of bacteria. REASON FOR DEVELOPING SHUTTLE VECTOR Prokaryotic vectors cannot exist & work in eukaryotic cells because the system of two groups of organisms varies. Therefore, vectors with two origin of replication were constructed which may exist in both eukaryotes and prokaryotes. Since these vectors can be grown in one host and then moved into another without any extra manipulation, they are called shuttle vectors. These vectors have been designed to replicate in cells of two different species; therefore, they contain two origins of replication, one specific for each host species, as well as those genes necessary for their replication and not provided by the host cells. These vectors are created by recombinant techniques. Some of them can be grown in two different prokaryotic species, usually E. coli and a eukaryotic one, e.g. yeast, plants, animals. YEp13 is an example of shuttle vector. The 2 µm plasmid is an excellent basis for a cloning vector. It is 6 kb in size, which is ideal for a vector, and exists in the yeast cell at a copy number of between 70 and 200. Replication makes use of a plasmid origin, several enzymes provided by the host cell, and the proteins coded by the REP1 and REP2 genes carried by the plasmid. However, all is not perfectly straightforward in using the 2 µm plasmid as a cloning vector. First, there is the question of a selectable marker. In order to use LEU2 as a selectable marker, a special kind of host organism is needed. Selection is possible because transformants contain a plasmid-borne copy of the LEU2 gene, and grow in the absence of the amino acid. In a cloning experiment, cells are plated out onto minimal medium, which contains no added amino acids. Only transformed cells are able to survive and form colonies. USES It might be difficult to recover the recombinant DNA molecule from a transformed yeast colony. This is not such a problem with YEps, which are present in yeast cells primarily as plasmids, but with other yeast vectors, which may integrate into one of the yeast chromosomes, purification might be impossible. This is a disadvantage because in many cloning experiments purification of recombinant DNA is essential in order for the correct construct to be identified by, for example, DNA sequencing. The standard procedure when cloning in yeast is therefore to perform the initial cloning experiment with E. coli, and to select recombinants in this organism.

1. Shuttle vector
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2. Vectors
 DNA molecule originating from a virus, a plasmid, or the cell of a
higher organism into which another DNA fragment of appropriate size
can be integrated without the loss of the vector capacity for self-
replication.
 Vectors introduce foreign DNA into the host cells, where it can be
reproduced in large quantities.

3. Shuttle vector definition
 A shuttle vector is a vector (usually a plasmid)
constructed so that it can propagate in two
different host species. It include plasmids that
can propagate in eukaryotes and prokaryotes or
in different species of bacteria

4. REASON FOR DEVELOPING SHUTTLE
VECTOR
 Prokaryotic vectors cannot exist & work in eukaryotic
cells because the system of two groups of organisms
varies.
 Therefore, vectors with two origin of replication were
constructed which may exist in both eukaryotes and
prokaryotes.

5. Shuttle vector
 Since these vectors can be grown in one host and then moved into
another without any extra manipulation, they are called shuttle vectors.
 These vectors have been designed to replicate in cells of two different
species; therefore, they contain two origins of replication, one specific
for each host species, as well as those genes necessary for their
replication and not provided by the host cells.
 These vectors are created by recombinant techniques.
 Some of them can be grown in two different prokaryotic species,
usually E. coli and a eukaryotic one, e.g. yeast, plants, animals.
 YEp13 is an example of shuttle vector.

6. Shuttle vector
 The 2 µm plasmid is an excellent basis for a cloning
vector. It is 6 kb in size, which is ideal for a vector, and
exists in the yeast cell at a copy number of between 70
and 200.
 Replication makes use of a plasmid origin, several
enzymes provided by the host cell, and the proteins
coded by the REP1 and REP2 genes carried by the
plasmid.

7. Shuttle vector
 However, all is not perfectly straightforward in using the 2
µm plasmid as a cloning vector. First, there is the question of
a selectable marker.
 In order to use LEU2 as a selectable marker, a special kind
of host organism is needed.

8. Shuttle vector
 Selection is possible because transformants contain a plasmid-
borne copy of the LEU2 gene, and grow in the absence of the
amino acid.
 In a cloning experiment, cells are plated out onto minimal
medium, which contains no added amino acids. Only
transformed cells are able to survive and form colonies.

10. USES OF SHUTTLE VECTOR
 It might be difficult to recover the recombinant DNA
molecule from a transformed yeast colony.

11. USES OF SHUTTLE VECTOR
 This is not such a problem with YEps, which are present in yeast
cells primarily as plasmids, but with other yeast vectors, which
may integrate into one of the yeast chromosomes, purification
might be impossible.
 This is a disadvantage because in many cloning experiments
purification of recombinant DNA is essential in order for the
correct construct to be identified by, for example, DNA
sequencing.
 The standard procedure when cloning in yeast is therefore to
perform the initial cloning experiment with E. coli, and to select
recombinants in this organism.

12. Expression Vector definition
 The expression vectors are vectors which act as
vehicles for DNA insert and also allow the DNA insert
to be expressed efficiently. These may be plasmids or
viruses. The expression vectors are also known as
expression constructs.

14. Expression Vector
 The expression vectors are genetically engineered for the introduction of
genes into the target cells. In addition to the gene of interest, these
expression constructs also contain regulatory elements like enhancers and
promoters so that efficient transcription of the gene of interest occurs.
 The simplest expression constructs are also known as transcription
vectors; only because they allow transcription of the cloned foreign gene
and not its translation. The vectors which facilitate both transcription and
translation of the cloned foreign gene are known as protein expression
vectors. These protein expression constructs also lead to the production of
recombinant protein.

15. How do Expression Vectors work?
 Once the expression construct is inside the host cell, the
protein encoded by the gene of interest is produced by
the transcription. Thereafter, it utilizes the translation
machinery and ribosomal complexes of the host
organism.
 Frequently, the plasmid is genetically engineered to
harbor regulatory elements like enhancers and
promoters. These regulator sequences aid in efficient
transcription of the gene of interest.

16. How do Expression Vectors work?
 Expression vectors are extensively used as tools which
help in the production of mRNAs and, in turn, stable
proteins. They are of much interest in biotechnology and
molecular biology for the production of proteins like
insulin. Insulin is the chief ingredient in the treatment of
the complex disease, Diabetes.
 When the protein product is expressed, it is to be then
purified. The purification of a protein poses a challenge
since the protein of interest, whose gene is carried on
the expression vector, is to be purified independently of
the proteins of the host organism.

17. Expression Vectors
 Therefore, expression vectors must have the following
expression signals:
 Strong promoter
 Strong termination codon
 Adjustment of distance between the promoter and
cloned gene
 Inserted transcription termination sequence

19. Uses
 Expression vectors are the basic tools in
biotechnology for the production of proteins.
Escherichia coli is commonly used as the host for
protein production, but other cell types may also
be used. An example of the use of expression
vector is the production of insulin, which is used
for medical treatments of diabetes.

20. Differences shuttle and expression
vectors
Expression vectors are used for molecular
biology techniques such as site-directed
mutagenesis.
 A shuttle vector is a vector that can
propagate in two different host species,
hence, inserted DNA can be tested or
manipulated in two different cell types