Transcript File - Future is BioTechnology.

Unit II Lecture 4
B. Tech. (Biotechnology) III Year
V th Semester
EBT-501, Genetic Engineering
Unit II
• Restriction modification
• Enzymes used in recombinant DNA technology
endonucleases, ligases and other enzymes useful in
gene cloning
• PCR technology for gene/DNA detection, cDNA,
• Use of Agrobacterium for genetic engineering in
plants
• Gene libraries; Use of marker genes.
• Cloning of foreign genes: DNA delivery methods physical methods and biological methods,
• Genetic transformation of prokaryotes: Transferring DNA
into E. coli –Chemical induction and Electroporation,
Gene Transfer in Plants
The Ti plasmid of Agrobacterium
tumefaciens is an important tool for
transferring genes into plants.
Agro-bacterium tumefacians
Agro bacterium tumefacians is a bacterium that
causes a disease known as crown gall in plants.
Infects plants by transferring its genetic material into
plant cell.
Agrobacterium transformation is the most common
technique for genetically engineered plants
Production of Transgenic Plants
• Microprojectile bombardment
• Electroporation
• Agrobacterium tumefaciens-mediated
transformation
• Plant cells are totipotent, so an entire plant can
be regenerated from a genetically engineered
plant somatic cell.
Agrobacterium tumifaciens
Infection
The Ti Plasmid
Transformation of Plant Cells by
the Ti Plasmid
Key steps from natural
Agrobacterium to “useful”
Agrobacterium
• Some vir genes deleted--disarmed
– Opines not going to be produced
– Deleting tumorogenesis function
• Choosing strains that transfer DNA in lab
• Clone in genes of interest, antibiotic
resistance genes, etc.
• Binary system-- two plasmids are better
than one Ti plasmid
Plant transformation with the Ti plasmid of
Agrobacterium tumefaciens
• Tumor formation is the result of the
transfer, integration and expression of
genes on a specific segment of A.
tumefaciens plasmid DNA called the TDNA (transferred DNA)
• The T-DNA resides on a large plasmid
called the Ti (tumor inducing) plasmid
found in A. tumefaciens
The Ti plasmid of Agrobacterium tumafaciens and
the transfer of its T-DNA to the plant nuclear
genome
Ti plasmid: structure & function
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The infection process:
Wounded plant cell releases
phenolics and nutrients.
Phenolics and nutrients cause
chemotaxic response of A.
tumefaciens
Attachment of the bacteria to the
plant cell.
Certain phenolics (e.g.,
acetosyringone,
hydroxyacetosyringone) induce
vir gene transcription and allow
for T-DNA transfer and
integration into plant
chromosomal DNA.
Transcription and translation of
the T-DNA in the plant cell to
produce opines (food) and
tumors (housing) for the
bacteria.
The opine permease/catabolism
genes on the Ti plasmid allow A.
tumefaciens to use opines as a
C, H, O, and N source.
The binary Ti plasmid system involves using a
small T-DNA plasmid (shown below) and a
disarmed (i.e., no T-DNA) Ti plasmid in A.
tumefaciens
DNA-delivery methods
Method
Comment
Ti plasmid-mediated gene
transfer *
Excellent and highly effective, but
limited to dicots
Microprojectile bombardment *
Easy and effective; used with a wide
range of plants
Viral vectors
Not very effective
Direct gene transfer into plant
protoplasts
Only certain protoplasts can be
regenerated into whole plants
Microinjetion
Tedious and slow
Electroporation *
Limited to protoplasts that can be
regenerated into whole plants
Liposome fusion
Limited to protoplasts that can be
regenerated into whole plants
Microprojectile bombardment or biolistic-mediated DNA
transfection equipment
• equipment
(a) lab version
(b) portable version
• When the helium pressure builds
to a certain point, the plastic
rupture disk bursts, and the
released gas accelerates the flying
disk with the DNA-coated gold
particles on its
• lower side. The gold particles pass
the stopping screen, which holds
back the flying disk, and penetrate
the cells of the plant.
Some plant cell reporter and selectable
marker gene systems
Enzyme activity
Selectable
marker
Reporter
gene
Neomycin phosphotransferase (kanr)
Yes
Yes
Hygromycin phosphotransferase (hygr)
Yes
Yes
Nopaline synthase
No
Yes
Octopine synthase
No
Yes
-glucuronidase (GUS)
No
Yes
Firefly luciferase
No
Yes
-galactosidase
No
Yes
Bromoxynil nitrilase
Yes
No
Green fluorescent protein (GFP)
No
Yes
Genetically Modified Organisms