A cloning vector is a DNA molecule that is used to carry a foreign DNA into the host cell. It has the ability to self replicate and integrate into the host cell.Saccharomyces cerevisiae. Development of plasmid is most commonly found in strains of S. cerevisiae its called 2μm plasmid present in Eukaryotic cells 6kb in size, high copy number 70-200 Replication depend on 1. plasmid origin 2.enzymes by host cell 3. proteins encoded by REP1 AND REP2 FLP gene –inversion of set of sequences within the plasmid Carry genes conferring resistance to inhibitors such as methotrexate,copper Ti PLASMID OF Agrobacterium tumefaciens -soil microorganism-causes crown gall disease -crown gall occurs as follows 1.Wound on a stem 2. A.tumefaciens invade the plant 3. infection- cancerous proliferation-at region of crown -Lipopolysaccharides(polygalactouronic) make phenolic compounds to induce vir gene-vir gene produce enzyme that produce ssDNA with nick on same strand-leads to opine synthesis(for proliferation of bacteria)-auxin and cytokinin- disorganised proliferation of cells-leads to crown gall - Ability to cause crown gall is ti plasmids-tumour inducing plasmid within bacterial cell -large plasmid, greater than 200kb, carries numerous genes invloved in infective process. -after infection part of molecule integrated to plant chromosomal DNA-segment T-DNA-15-30kb in size -maintained as a stable form in plant cell-passed on to daughter cells -phenolic compounds released are acetosyringone and hydroxysyringone -vir genes-35 kb-contains site for opine synthesis and opine catabolism -2 main strategies involved in inserting new DNA molecule into plasmid 1. BINARY VECTOR STRATEGY 2.CO INTEGRATIVE STRATEGY

1. CLONING VECTOR FOR
YEAST,
PLANTS(AGROBACTERIUM)

2. Generally what is a cloning vector??
2
▸ A cloning vector is a DNA molecule that is used
to carry a foreign DNA into the host cell.
▸ It has the ability to self replicate and integrate
into the host cell.

3. Y WAITING LETS GO
DEEP INTO IT
Today lets see about
*cloning vectors (yeast)
*cloning
vectors(Agrobacterium
tumefaciens)
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4. CLONING VECTORS FOR YEAST
▸ Saccharomyces cerevisae
▸ Development of plasmid is most
commonly found in strains of S.
cerevisiae
▸ its called 2μm plasmid present in
Eukaryotic cells
▸ 6kb in size, high copy number 70-200
▸ Replication depend on
▸ 1. plasmid origin
▸ 2.enzymes by host cell
▸ 3. proteins encoded by REP1 AND REP2
▸ FLP gene –inversion of set of
sequences within the plasmid
▸ Carry genes conferring resistance to
inhibitors such as
methotrexate,copper
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5. “
Types of yeast cloning vectors
1. Yeast episomal plasmids
2. Yeast replicative plasmids
3. Yeast integrative plasmid
Lets have a quick brief about
the types……….
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6. TYPES OF YEAST CLONING VECTORS
1.YEAST EPISOMAL PLASMIDS
-size 10.7 kb
-derived from 2μm plasmid (eg., YEP 13)
-include entire pBR322 sequence
-shuttle vector(replicate in yeast and E.Coli)
-selectable marker LEU2 gene, ampRand tetR antibiotic resistance
genes
2.Yeast integrative plasmids
-basically bacterial plasmids, carry yeast gene (eg., YIP5)
-Its pBR322 with inserted URA3 gene- codes for orotidine-5’-phosphate
-Also used as selectable marker as LEU2 gene
-no parts of 2μm plasmid, cant replicate independently, survival by
integrating with yeast chromosomal DNA
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7. 3.YEAST REPLICATIVE
PLASMIDS (eg.,YRP7)
-multiply as independent
plasmids, carry ori
-1 or 2 selectable markers
-(YRP7= pBR322+yeast gene
TRP1)- Involved in tryptophan
biosynthesis
- TRP1 has its own origin of
replication to replicate
TYPES OF YEAST CLONING VECTORS
YEAST ARTIFICIAL CHROMOSOME (YAC)
-upto 200kb can be cloned
-cloning inside yeast eukaryotic cell
-possessyeast telomere and yeast centromere
-production of gene libraries,
-Sizeof YAC-11.4kb
-special components- TRP1, URA3, TRP1-ori-cen4
region, TEL, SUP4
-Physical mapsof large genomes can be
constructed
- Transformation efficiency, yield of clones DNA
is less
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8. ENOUGH
WITH
CLONING
VECTORS
OF
YEAST….
LETS MOVE TO
NEXT INTERESTING
TOPIC …CLONING
VECTORS OF
PLANTS
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9. CLONING VECTORS FOR PLANT
Ti PLASMID OF Agrobacterium tumefaciens
-soil microorganism-causes crown gall disease
-crown gall occurs as follows
1.Wound on a stem
2. A.tumefaciens invade the plant
3. infection- cancerous proliferation-at region
of crown
-Lipopolysaccharides(polygalactouronic) make
phenolic compounds to induce vir gene-vir
gene produce enzyme that produce ssDNA
with nick on same strand-leads to opine
synthesis(for proliferation of bacteria)-auxin
and cytokinin- disorganised proliferation of
cells-leads to crown gall
- Ability to cause crown gall is ti plasmids-
tumour inducing plasmid within bacterial cell
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10. Ti plasmid
-large plasmid, greater than 200kb, carries numerous genes
invloved in infective process.
-after infection part of molecule integrated to plant
chromosomal DNA-segment T-DNA-15-30kb in size
-maintained as a stable form in plant cell-passed on to
daughter cells
-phenolic compounds released are acetosyringone and
hydroxysyringone
-vir genes-35 kb-contains site for opine synthesis and opine
catabolism
-2 main strategies involved in inserting new DNA molecule into
plasmid
1. BINARY VECTOR STRATEGY 2.CO INTEGRATIVE STRATEGY
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11. BINARY VECTOR
STRATEGY
-based on the observation that the T-DNA
does not need to be physically attached to the
rest of the Ti plasmid.
-A two-plasmid system, with the T-DNA on a
relatively small molecule, and the rest of the
plasmid in normal form, is just as effective at
transforming plant cells.
-In fact, some strains of A. tumefaciens, and
related agrobacteria, have natural binary
plasmid systems.
- The T-DNA plasmid is small enough to have a
unique restriction site and to be manipulated
using standard techniques.
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12. CO INTEGRATIVE
VECTOR STRATEGY
-usesan entirely new plasmid, based on an E. coli
vector, but carrying a small portion of the T-DNA.
-The homology between the new molecule and the Ti
plasmid means that if both are present in the sameA.
tumefaciens cell, recombination can integrate the E.
coli plasmid into the T-DNA region.
-The gene to be cloned is therefore inserted into a
unique restriction site on the small E. coli plasmid,
introduced into A. tumefaciens cells carrying a Ti
plasmid, and the natural recombination process left to
integratethe new gene into the T-DNA.
- Infection of the plant leadsto insertion of the new
gene, along with the rest of the T-DNA, into the plant
chromosomes.
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