EXPRESSION VECTOR,

PLASMID VECTOR

Recombinant Technology

a vector is a DNA molecule used as a vehicle to transfer foreign

genetic material into another cell.

The four major types: Plasmids, Bacteriophages and other

viruses, Cosmids, and Artificial chromosomes.

Common to all engineered vectors are an origin of replication, a

multicloning site, and a selectable marker.

The purpose of a vector which transfers genetic information to

another cell is typically to isolate, multiply, or express the insert in

the target cell. expression vectors specifically are for the expression

of the transgene in the target cell, and generally have a promoter

sequence that drives expression of the transgene.

Insertion of a vector into the target cell is generally called

transfection, although insertion of a viral vector is often called

transduction.

An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell.

Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery.

The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.

The goal of a well-designed expression vector is the production of large amounts of stable messenger RNA.

Expression vectors require not only transcription but translation of the vector's insert, thus requiring more components than simpler transcription-only vectors. Expression vectors require sequences that encode for: Polyadenylation tail: Creates a polyadenylation tail at the

end of the transcribed pre-mRNA that protects the mRNA from exonucleases and ensures transcriptional and translational termination: stabilizes mRNA production.

Minimal UTR length: UTRs contain specific characteristics that may impede transcription or translation, and thus the shortest UTRs or none at all are encoded for in optimal expression vectors.

Kozak sequence: Vectors should encode for a Kozaksequence in the mRNA, which assembles the ribosome for translation of the mRNA.

After expression of the gene product, the

purification of the protein is required; but since

the vector is introduced to a host cell, the

protein of interest should be purified from the

proteins of the host cell. Therefore, to make

the purification process easy, the cloned gene

should have a tag. This tag could be histidine

(His) tag or any other marker peptide.

DNA vectors that are used in many molecular-biology gene-cloning experiments need not result in the expression of a protein. Expression vectors are basic tools for biotechnology and the production of proteins such as insulin that are important for medical treatments of specific diseases like diabetes.

Expression vectors must have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).

Eco R1

Cla 1

pYE4

Trp 1

Plasmid vectors

A plasmid is an extra-chromosomal DNA molecule separate from the

chromosomal DNA which is capable of replicating independently of the

chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids

usually occur naturally in bacteria, but are sometimes found in eukaryotic

organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).

Plasmids used in genetic engineering are called plasmid vectors

The plasmids are inserted into bacteria by a process called transformation.

Then, the bacteria are exposed to the particular antibiotics.

Only bacteria which take up copies of the plasmid survive , since the plasmid makes them resistant.

Now these bacteria can be grown in large amounts, harvested and lysed to isolate the plasmid of interest.

However, a plasmid can only contain inserts of about 1–10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used.

There are

two types of

plasmid

integration into

a host bacteria:

Non-integrating

plasmids

replicate

Integrate into

the host

Derived from E. coli plasmid ColE1), which is 4,362 bp DNA and was

derived by several alterations in earlier cloning vectors.

pBR322 is named after Bolivar and Rodriguez, who prepared this vector.

It has genes for resistance against two antibiotics (tetracycline and

ampicillin), an origin of replication and a variety of restriction sites for

cloning of restriction fragments obtained through cleavage with a specific

restriction enzyme.

It has unique restriction sites for 20 restriction endonucleases.

Certain restriction sites for eg., BAM HI in the tetr genes of the plasmid are

present within the gene in such a way that the insertion of foreign segment

of DNA will inactivate the tetr gene.

The recombinant plasmid will allow the cells to grow only in the presence of

ampicillin but will not protect them against tetracyclin.

Thus recombinant plasmids selection will be easily carried out.

Another series of plasmids that are used as cloning vectors belong to pUC

series (after the place of their initial preparation I.e. University of California).

These plasmids are 2,700 bp long and possess

Ampicillin resistance gene

The origin of replication derived from pBR322 and

The lacz gene derived from E.coli.

Within the lac region is also found a polylinker sequence having unique

restriction sites.

When DNA fragments are cloned in this region of pUC, the lac gene is

inactivated.

These plasmids when transformed into an appropriate E. coli strain, which is

lac (JM103, JM109), and grown in the presence of IPTG (isopropyl

thiogalactosidase, which behaves like lactose, and induced the synthesis of

b-galactosidase enzyme) and X-gal (substrate for the enzyme), will give rise

to white or clear colonies.

On the other hand, pUC having no inserts are transformed into bacteria, it

will have active lac Z gene and therefore will produce blue colonies, thus

permitting identification of colonies having pUC vector with cloned DNA

segments.

The cloning vectors belonging to pUC family are available in pairs with

reverse orders of restriction sites relative to lac Z promoter. pUC8 and pUC9

are one such pairs.

Other similar pairs include

pUC12 and pUC13 and

pUC18 and pUC19.

Hind II

PstISal I

BamHI

Sma I

EcoRI

EcoRI

Sma I

BamHISal I

PstI

Hind II

pUC8 (2768 bp pUC9 (2768 bp

B-galactosidase gene

pUC

Ti Plasmid

The most commonly used vector for gene transfer in

higher plants are based on tumour inducing mechanism

of the soil bacterium A. tumefaciens, which is the causal

organism for crown gall disease.

The disease is caused due to transfer of DNA segment

from the bacterium to the plant nuclear genome.

The DNA segment which is transferred is called T-DNA

and is part of a large Ti plasmid found in virulent strains

of A. tumefaciens.

Most Ti plasmids have four regions in common

Region A: comprising T-DNA which is responsible for tumour induction leading to

production of tumors with altered morphology (shooty or rooty mutant galls).

Sequences homologous to this region are always transferred to plant nuclear

genome, so that the region is described as T-DNA (transferred DNA)

Region B: This region is responsible for replication

Region C Responsible for conjugation

Region D: Responsible for virulence, so that mutation in this region abolishes

virulence. This region is therefore called virulence (vir) region and plays a crucial

role in the transfer of T-DNA into the plant nuclear genome.

The T-DNA consist of following regions:

An ONC region: Consisting of 3genes (2 genes tms1 and tms2

rep shooty locus and one gene tmr rep rooty locus) responsible

for 2 phytohormones, namely IAA and isopentyladenosine-5’-

monophosphate (a cytokinin). These genes encode the enzymes

responsible for the synthesis of these phytohormones so that the

incorporation of these genes in plant nuclear genome leads to the

synthesis of phytohormones in the host plant. These

phytohormones in turn alter the development programme,

leading to the formation of crown gall.

An OS region: responsible for synthesis of opines.

These are derivatives of various compounds (eg. Arginine +

pyruvate) that are found in plant cells.

Two most common opines are octopine and nopaline.

The enzymes for sytnhesis of these 2 opines, are encoded by

(octopine synthase and nopaline synthase) T-DNA.

Depending upon the opine it codes, they are described as octopine

type Ti plasmid or nopaline type Ti plasmid.

The T-DNA region in the plasmid is flanked on both

the sides with 25 bp direct repeat sequences, which are

essential for T-DNA transfer, acting only in its cis

orientation.

Any DNA sequences flanked by these 25 bp repeated

sequence in the correct orientation, can be transferred

to plant cells.

This is successfully utilized for Agrobacterium

mediated gene transfer in higher plants.

http://en.wikipedia.org/wiki/Expression_vector

http://en.wikipedia.org/wiki/Vector_(molecular_biology)

http://www.plantmethods.com/content/1/1/13

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/i

n-vitro-genetics/expression-vectors.html

http://en.wikipedia.org/wiki/Plasmid

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