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Vectors

◘ we will taking about 3 Vectors in our lecture : -

1- Cosmid Vector .

2- Yeast artificial chromosome Vector .

3- Plant Vector .

Cosmid Vector

• A cosmid is a type of hybrid plasmid that contains a Lambda

phage cos sequence, (cos sites + plasmid = cosmids)

• Host cell : ( Bacteria ) Prokaryotic (E. coli host )

• DNA sequences are originally from the lambda phage.

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

• Cosmids can contain 44 k bp of DNA.

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• They also contain a gene for selection such as antibiotic resistance, so

that the transformed cells can be identified by plating on a medium

containing the antibiotic. Those cells which did not take up the cosmid

would be unable to grow.

• They Have Multiple cloning site ( poly linker ) , and Origin of

replication .

• Extra-chromosomal circular DNA molecule .

◘ Cloning Steps :-

1- Preparation .

- Target DNA: the genomic DNA to be cloned has to be cut into the

appropriate size range of restriction fragments , By using Restriction

Enzymes , And Cut the Cosmid Cloning site ( poly linker ) by the same

Restriction enzyme , Then Ligation of physically unlinked fragments.

2- Packaging

- the total DNA is transferred into an appropriate E. coli host via a

technique called in vitro packaging (recombinant plasmids contained in

phage shells ) .

- they can also be packaged in phage capsids, which allows the foreign

genes to be transferred into or between cells by transduction (a process

resembling viral infection )

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3- Plating & Growing & Selection & Isolation

- Colonies are formed in selective media , just as with a plasmid vector .

- The media is : Liquid media .

- So that the transformed cells can be identified by plating on a medium

containing the antibiotic ( Ampicillin Agar plate ).

- Those cells which did not take up the cosmid would be unable to grow.

Yeast artificial chromosome Vector

Telomers

AB Resistant Centromere

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- Yeast artificial chromosome (YAC) is a human-engineered

DNA molecule used to clone DNA sequences in yeast cells.

- YAC is an artificially constructed chromosome that contains a

Centromere :

Telomeres :

• for maintenance and stabilization and protect ends from degradation

happened by enzymes .

Autonomous replicating sequence (ARS)

• element required for replication and preservation of YAC

in yeast cells

ARS elements are thought to act as replication origins

- Artificial chromosome that contains telomers , (center ) origin

of replication , a yeast centromere , and selectable marker (

Antibody resistance ) for identification in yeast cells.

- Host cell : Yeast cell ( Eukaryotic )

◘ A YAC is built using an initial circular plasmid

- typically broken into two linear molecules using restriction

enzymes

DNA ligase is then used to ligate a sequence or gene of

interest between the two linear molecules

forming a single large linear piece of DNA

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Plant Vectors

1- Ti – plasmid ( 1ry vector )

2- plant vector ( 2ry vector )

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◘ Ti – Plasmid

Tumor inducing plasmids (Ti plasmid) are double stranded

circular DNA present in Agrobacterium tumefaciens.

Agrobacterium is a Gram negative soil bacterium which

infects plants. ( Host cell : plant cell )

Agrobacterium tumefaciens infects damaged plant tissues,

induces the formation of a plant tumor growth called crown

gall ( grown gall ) .

Crown gall ( grown gall ) formation occurs when a fragment

of Ti plasmid, referred to as ( T-DNA ) is transferred from

the bacterium in to the host where it gets integrated in to the

plant cell chromosomes.

◘ Plant Vector : -

- The gene of interest integrate with the DNA of the plant ,

produce new traits for the plant using later in Genetic

engineering .

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