Submitted to:Submitted to: Submitted by:Submitted by: Dr. Anita grewal & ABHISHEK DHIMANDr. Anita grewal & ABHISHEK DHIMANDr. Sunita khatak biotech – 6Dr. Sunita khatak biotech – 6thth sem sem 2507457 2507457

Bacteria origin vectorsBacteria origin vectors

VECTORVECTOR

Vector is a DNA molecule that has the ability to Vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host replicate autonomously in an appropriate host cell.cell.

For the DNA fragment to be cloned (called dna For the DNA fragment to be cloned (called dna insert) is integrated for cloning.insert) is integrated for cloning.

Vector must have origin of dna replication.Vector must have origin of dna replication.

Properties of a good vectorProperties of a good vector1.1. It should be replicate autonomously.It should be replicate autonomously.

2.2. A vector should be ideally less than 10 kb in size.A vector should be ideally less than 10 kb in size.

3.3. Vector should be easy to isolate and purify.Vector should be easy to isolate and purify.

4.4. The vector should have suitable markers genes for The vector should have suitable markers genes for detection and selection.detection and selection.

5.5. A vector should contain unique target sites for the RE by A vector should contain unique target sites for the RE by which the dna insert can be integrated without disrupting which the dna insert can be integrated without disrupting an essential function.an essential function.

Different types of bacteria origin vectors are:Different types of bacteria origin vectors are:

a. Plasmid vector :a. Plasmid vector :

1.1. Pbr322 vectorPbr322 vector2.2. Plasmid pUC18 vectorPlasmid pUC18 vector3.3. pSC101 vectorpSC101 vector

b. Bacteriophage vectorb. Bacteriophage vector

c. Cosmid vectorc. Cosmid vector

d. Fosmid vectord. Fosmid vector

e. Shuttle vectore. Shuttle vector

An ideal pBR322 have the following functions:

Minimum amount of dna less than 10kb.

At least two selectable markers.

Only one recognition site at least one restriction endonuclease.

pBR322 is one of the most popular and most widely used plasmid vector of 4363 bp.

It has two selectable markers (tetracycline, tet r and ampicillin, amp r resistance gene) and only single or unique recognition sites for different 12 restriction enzymes (PstI, SacI, PvuI located with Amp r gene and BamHI, SalI,etc. within tet r gene).

Plasmid pUC18Plasmid pUC18 Derivatives of pBR322 and much smaller (size upto 2.7 kb)Derivatives of pBR322 and much smaller (size upto 2.7 kb)

Naturally occuring plasmid are viruses of bacteria.Naturally occuring plasmid are viruses of bacteria.

Artificial plasmid pUC18 has been genetically engineered to Artificial plasmid pUC18 has been genetically engineered to include a gene for antibiotic resistance to ampicillin ,and a include a gene for antibiotic resistance to ampicillin ,and a lacZ gene for the enzyme beta-galactosidase.lacZ gene for the enzyme beta-galactosidase.

lacZ gene contains a polylinker with series of unique lacZ gene contains a polylinker with series of unique restriction sites.restriction sites.

Digestion by the endonuclease will maka a single cut and Digestion by the endonuclease will maka a single cut and allow the circular plasmid dna to recombine with foreign allow the circular plasmid dna to recombine with foreign dna.dna.

pSC101vectorpSC101vector Plasmid vector contains replication module(ori) for Plasmid vector contains replication module(ori) for

replication in E.coli ,tet r gene ,HindIII, BamHI,SalI.replication in E.coli ,tet r gene ,HindIII, BamHI,SalI.

Insertion od dna fragement into the recognition sites Insertion od dna fragement into the recognition sites disrupts the tet r gene.disrupts the tet r gene.

Cells transformed by it are sensitive to the tetracycline and Cells transformed by it are sensitive to the tetracycline and are distinguish easily to resistant to antibiotic.are distinguish easily to resistant to antibiotic.

Vector maintained the 5 copies per cell and the multiple Vector maintained the 5 copies per cell and the multiple cloning sites.cloning sites.

It is a gram positive bacteria and allows insertion upto (6-It is a gram positive bacteria and allows insertion upto (6-12kb).12kb).

Lambda phage vectorsLambda phage vectors Fragments up to 23 kb can be may be accommodated by a phage vectorFragments up to 23 kb can be may be accommodated by a phage vector

Lambda is most common phageLambda is most common phage

60% of the genome is needed for lytic pathway.60% of the genome is needed for lytic pathway.

Segments of the Lambda DNA is removed and a stuffer fragment is put in.Segments of the Lambda DNA is removed and a stuffer fragment is put in.

The stuffer fragment keeps the vector at a correct size and carries marker The stuffer fragment keeps the vector at a correct size and carries marker genes that are removed when foreign DNA is inserted into the vector.genes that are removed when foreign DNA is inserted into the vector.

Example: Charon 4A LambdaExample: Charon 4A Lambda

When Charon 4A Lambda is intact, beta-galactosidase reacts with X-gal When Charon 4A Lambda is intact, beta-galactosidase reacts with X-gal and the colonies turn blue.and the colonies turn blue.

When the DNA segment replaces the stuffer region, the lac5 gene is When the DNA segment replaces the stuffer region, the lac5 gene is missing, which codes for beta-galactosidase, no beta-galactosidase is missing, which codes for beta-galactosidase, no beta-galactosidase is formed, and the colonies are white.formed, and the colonies are white.

Cosmid vectorsCosmid vectors Cosmids are the essentially plasmids of minimum of 250 bp Cosmids are the essentially plasmids of minimum of 250 bp

of of λλ DNA DNA..

A typical cosmid has replication origin, unique restriction A typical cosmid has replication origin, unique restriction sites and selectable markers.sites and selectable markers.

For long dna inserts between two cos sites frm 38 to 52 kb.For long dna inserts between two cos sites frm 38 to 52 kb.

DNA fragment used for cloning are produced by partial DNA fragment used for cloning are produced by partial digestion with a RE.digestion with a RE.

Cosmids can maximum accomodate upto 40 kb long.Cosmids can maximum accomodate upto 40 kb long.

Can packaged into lambda particles & selection for the Can packaged into lambda particles & selection for the recombinant DNA.recombinant DNA.

pJB8 cosmid vectorpJB8 cosmid vector

Fosmid vectorsFosmid vectors A useful derivative of BAC vectors is called fosmid.A useful derivative of BAC vectors is called fosmid.

A fosmid vector is in essence a bac vector that contains A fosmid vector is in essence a bac vector that contains lambda phage cos sites to facilitate its packaging.lambda phage cos sites to facilitate its packaging.

Example of a fosmid vector is a pFOS1 which produced by Example of a fosmid vector is a pFOS1 which produced by in vivo homologus recombination between two cos sites.in vivo homologus recombination between two cos sites.

It has two cos sites.It has two cos sites.

Fosmids are the essentially mini-bacs that are used to clone Fosmids are the essentially mini-bacs that are used to clone of dna inserts upto 40 kb.of dna inserts upto 40 kb.

Shuttle vectorsShuttle vectors Shuttle vector is that which can propagate in two different host Shuttle vector is that which can propagate in two different host

species.species.

Main advantage of these vectors is can be manipulated in E.coli.Main advantage of these vectors is can be manipulated in E.coli.

Shuttle vectors include plasmids can propagate in eukaryotes and Shuttle vectors include plasmids can propagate in eukaryotes and prokaryotes .prokaryotes .

They are frequently used to make multiple copies of gene in E.coli.They are frequently used to make multiple copies of gene in E.coli.

They have the origin of replication and a selectable marker e.g They have the origin of replication and a selectable marker e.g antibiotic resistance ,antibiotic resistance ,ββ lactamase. lactamase.

Autonomously replicating sequence, a yeast centromere and yeast Autonomously replicating sequence, a yeast centromere and yeast selectable marker.selectable marker.

Artificial chromosome vectorsArtificial chromosome vectors

Circular or linear vectors that are stably Circular or linear vectors that are stably maintained , 1 to 2 copy per cellmaintained , 1 to 2 copy per cell

There are several type of such vectors:There are several type of such vectors:

1.1. BAC vectorBAC vector

2.2. PAC vectorPAC vector

3.3. YAC vectorYAC vector

4.4. MAC vectorMAC vector

5.5. HAC vectorHAC vector

Bacterial artificial vectorsBacterial artificial vectors

BAC vectors have the origin of replication (oriS) of BAC vectors have the origin of replication (oriS) of E.coli f factor.E.coli f factor.

Low copy number of BAC’c help maintain the dna Low copy number of BAC’c help maintain the dna inserts without any change in it.inserts without any change in it.

First BAC vector was pBAC108L other BAC vector First BAC vector was pBAC108L other BAC vector pBeloBAC11.pBeloBAC11.

Size upto 7.4kb.Size upto 7.4kb.

Vector maintained in E.coli cells at single copy per Vector maintained in E.coli cells at single copy per cell.cell.

oriS, origin of replication, repE encodes to protein oriS, origin of replication, repE encodes to protein that binds oriS.that binds oriS.

parA and parB encode the protein that bind the parA and parB encode the protein that bind the site, parC ensure regular partioning of the vector.site, parC ensure regular partioning of the vector.

lacZ gene t7 and sp6 polymerase driven lacZ gene t7 and sp6 polymerase driven promoters.promoters.

Used to clone upto 300kb.Used to clone upto 300kb.

Extensively used in analysis of genomes, Extensively used in analysis of genomes, including the human genome.including the human genome.

Yeast Artificial ChromosomesYeast Artificial Chromosomes YACs can hold up to 500 kbs.YACs can hold up to 500 kbs.

YACs are designed to replicate as plasmids in bacteria when no foreign YACs are designed to replicate as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes.they then replicate as eukaryotic chromosomes.

YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers.of replication, and bacterial selectable markers.

YAC plasmidYAC plasmidYeast chromosomeYeast chromosome

DNA is inserted to a unique restriction site, and cleaves the plasmid with DNA is inserted to a unique restriction site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear.causes the YAC to become linear.

Once in the cell, the rYAC replicates as a chromosome, also replicating Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.the foreign DNA.