Genetic Engineering

1. Agrobacterium tumefaciens
BASED VECTORS
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Lecture- 8

2. Introduction
 Agrobacterium tumefaciens is capable of
transforming a plant cell naturally and has been
exploited to introduce DNA into plants.
 The pTi plasmid of Agrobacterium has a natural
tendency to catalyze the natural transfer of T-
DNA to plants.
 Exogenous DNA inserted into T-DNA is
naturally transferred to plants
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3. Tumour inducing plasmid pTi
Its size is greater than 200 kbp
T-DNA from pTi is transferred to the wounded cell
T-DNA is 23 kbp in size and flanked by 25 bp direct
repeats namely right border and left border.
Wild typeT-DNAs contains shoot inducing loci (shi) and
root inducing loci (roi) which comprise of genes
encoding for the synthesis of plant growth regulators
T-DNA encodes enzymes for the synthesis of novel
amino acid derivatives called opines which act as a
source of energy
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4. Location of useful genes and
sites on pTi plasmid
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5.  Processing and transfer of T-DNA is stimulated
by the products of vir genes
 pTi is capable of autonomous replication using
its own origin of replication
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6.  Tumour formation in plants occurs due toT-DNA
integration
 Disarming ofT-DNA is done to prevent oncogenicity
 While growing A. tumefaciens under in vitro conditions,
opine synthesizing and catabolizing genes can be
deleted from pTi
 Nitrogen and carbon sources can be supplied in the
medium
 For the transfer ofT-DNA, vir genes need not to be
present on the same vector and can be present in trans.
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7. pTi based Vectors
 pTi based vectors require manipulation of wild type
pTi in following respects:
 Size reduction
i)By disarming
ii)By deleting opine synthesizing and catabolising
genes
 Ability to replicate in E.coli:
i)pTi can not replicate in E.coli, so origin of
replication that can be used in E. coli must be added.
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8.  Insertion of selectable marker gene
 Insertion of polylinker / Multiple cloning site (MCS) is
added to facilitate insertion of cloned gene into the
region betweenT-DNA border sequences
 Deletion of tra loci to prevent conjugative ability
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9. Cointegrate plant
transformation vector
 Depending upon the trans and cis supply of vir gene products
two vector systems have been developed i.e. cointegrate
vector system and binary vector system
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10.  co-integrated vectors were among the first types of
modified and engineeredTi plasmids devised for
Agrobacterium -mediated transformation, but are not
widely used today.
 These vectors are constructed by homologous
recombination of an E. coli plasmid with theT-DNA
region of an endogenousTi plasmid in Agrobacterium.
 Integration of the two plasmids requires a region of
homology present in both.
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11. Co-integrated plasmid assembled by in
vitro manipulation normally contains:
 the vir genes
 the left and rightT-DNA borders
 an exogenous DNA sequence between the two
T-DNA borders
 plant and bacterial selectable markers
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12. Drawbacks of co-integrated
vectors
Co-integrated vectors in general are less popular
due to:
 long homologies required between theTi plasmid
and the E. coli plasmids making them difficult to
engineer and use
 Relatively inefficient gene transfer compared to the
binary vectors
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13. Binary Plant Transformation Vector
In the binary vector system, the two different plasmids
employed are:
A wide-host-range small replicon, called mini Ti plasmid
which has an origin of replication (ori) that permits the
maintenance of the plasmid in a wide range of bacteria
including E. coli and Agrobacterium. This plasmid typically
contains:
 foreign DNA in place of T-DNA
 the left and right T-DNA borders (or at least the right T-
border)
 markers for selection and maintenance in both E. coli and A.
tumefaciens
 a selectable marker for plants
 The plasmid is "disarmed", since its tumor-inducing genes
located in the T-DNA have been removed 13

14.  A helper Ti plasmid, harboured in A. tumefaciens, which lacks the
entire T-DNA region but contains an intact vir region.
In general, the transformation procedure is as follows:
 The recombinant small replicon is transferred via bacterial
conjugation or direct transfer to A. tumefaciens harboring a helper Ti
plasmid
 The plant cells are co-cultivated with the Agrobacterium to allow
transfer of recombinant T-DNA into the plant genome
 Transformed plant cells are selected under appropriate conditions
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15. 15

16. Examples of some commonly used
mini-Ti vectors
pBIN19 vector :
 11777 bp in size
 Has kanamycin resistance gene
 Has 8 restriction sites
 Has low copy number
pBI101 vector:
 It is a promoterless vector
 Has expression cassettes; one for nptII other for
gus gene 16