cosmid vector and yeast artificial chromosome vector and plant vectors ( ti – plasmid )
Post on 29-Jan-2018
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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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