Cloning Vector

Sidra AyyazRabia TariqKhadija RasheedShafaq Shahzad

GROUP MEMBERS

CLONING VECTOR

CLONING VECTOR

INTRODUCTIONA cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

FEATURES OF A CLONING VECTOR

Origin of replication. Cloning site. Selectable marker. Reporter gene.

TYPES OF CLONING VECTORS

1. Plasmid.

2. Bacteriophage.

3. Cosmid.

4. Bacterial Artificial Chromosome.

5. Yeast Artificial Chromosome.

PLASMID VECTOR

Plasmid vector is a small, piece of circular DNA found outside of the bacterial chromosome.

Capable of autonomous replication.

Can transfer genes from one cell to other.

Contains an origin of replication, allowing for replication independent of host’s genome.

Contains Selective markers for the selection of cells containing a plasmid.

Contains a Multiple Cloning Site (MCS). Easy to be isolated from the host cell. E.g pBR322, pUC19.

Plasmid Vectors

Properties Of Plasmid Vectors

Smaller plasmid vectors are preferred for many reasons:

The efficiency of transformation is inversely related to the size of the plasmid.

Larger plasmids are more difficult to characterize by restriction mapping.

The yield of foreign DNA is reduced with larger plasmids because these plasmids

replicate to lower copy numbers

Multiplication of Plasmids

Varieties Of Plasmids Based On Functions

Fertility F-plasmids which contain tra genes. They are capable of conjugation and result in the expression of sex pilli.

Resistance (R) plasmids which contain genes that provide resistance against antibiotics or poisons.

Col plasmids which contain genes that code for bacteriocins, proteins that can kill other bacteria.

Degradative plasmids which enable the digestion of unusual substances, e.g. toluene and salicylic acid.

Virulence plasmids which turn the bacterium into a pathogen.

Uses Of Plasmid Major use of plasmids is to make large

amounts of proteins.  Plasmid may also be used for gene transfer

into human cells as potential treatment in gene therapy so that it may express the protein that is lacking in the cells.

BACTERIOPHAGE VECTOR

Cloning Vector that uses a Bacteriophage as a means for making and storing exact copies of segments of DNA.

It infects bacteria.

TYPES The bacteriophages used for cloning are ;

i.  phage λ 

ii. M13 phage.

 Phage λ  Vector

Infect E.coli. Origin of replication. Size is 48,502 bp. High transformation efficiency,

about 1000 times more efficient than the plasmid vector.

Enterobacteria is important in the study of specialized transduction.

Types of Phage λ  Vector

a. Insertion vectors: contain a unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted. e.g  λgt10 and λZAPII.

b. Replacement vectors: replacement vector has two recognition sites for the restriction endonuclease used for cloning. e.g λWES.λB' and λEMBL4

DNA Cloning Using Phages As Vectors

Phage M13 Vector

A gene for the lac repressor. The operator region of

the lac Z gene. A lac promoter upstream of

the lac Z gene. A polylinker region.

Types of M13 vector

M13 mp1  form by introduction of the lacZ' genes into the m13 vector.

M13 mp2 has a slightly altered lacZ' gene.It is the simplest M13 cloning vector.

M13 mp7 form by the introduction of additional restriction sites into the lacZ' gene. The polylinker is inserted into the EcoRI site of M13mp2, to give M13mp7.

Complex M13 vectors have more complex polylinkers inserted into the lacz' gene, ability to take DNA fragments with two different sticky ends.

M13mp9 having same polylinker as in m13 mp8 but in the reverse orientation which is important in DNA sequencing.

Use Of M13 Vector

M13 vector use in nanostructures and nanotechnology.

COSMID VECTOR Hybrids between a phage DNA molecule and

a bacterial plasmid. An origin of replication (ori). A cos site . An ampicillin resistance gene (amp).

Restriction sites for cloning .

Cosmids can carry up to 50 kb of inserted DNA.

Uses Of Cosmids Cosmids can be used to build genomic

libraries.

They are often used as a cloning vector in genetic engineering.

Cloning By Using Cosmid Vectors

Bacterial Artificial Chromosome

DNA construct, based on a functional F-plasmid. F-plasmids play a crucial role because they

contain partition genes that promote the even distribution of plasmids after bacterial cell division.

Bacterial artificial chromosome's usual insert size is 150-350 kbp.

Like other vectors, BACs contain:

1. Origin (ori) sequence derived from an E. coli plasmid called the F factor.

2. Multiple cloning sites (restriction sites).

3. Selectable markers (antibiotic resistance)

Uses Of BAC Useful for sequencing large stretches of

chromosomal DNA.

Frequently used in genome sequencing projects.

YEAST ARTIFICIAL CHROMOSOME 

YAC are genetically engineered chromosomes derived from the DNA of the yeast.

Capable of carrying inserts of 100 - 1000 kbp. A YAC can be considered as a functional

artificial chromosome since it includes three specific DNA sequences:

TEL: Telomere located at each chromosome end, protects the linear DNA from degradation by nucleases.

CEN: Centromere which is the attachment site for mitotic spindle fibers, "pulls" one copy of each duplicated chromosome into each new daughter cell.

ORI: Replication origin sequences which are specific DNA sequences.

It also contains few other specific sequences like:

a. Selectable markers: that allow the easy isolation of yeast cells that have taken up the

artificial chromosome.

b. Recognition site: for the two restriction enzymes EcoRI and BamHI.

Uses Of YAC Used to express eukaryotic proteins that

require posttranslational modification.

Used for detailed mapping of specific regions of the genome.