Unit II Lecture 4

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• Unit II Lecture 4
• B. Tech. (Biotechnology) III Year V th Semester
• EBT-501, Genetic Engineering

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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,

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• Gene Transfer in Plants
• The Ti plasmid of Agrobacterium tumefaciens is an
  important tool for transferring genes into
  plants.

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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
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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.

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Agrobacterium tumifaciens Infection
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The Ti Plasmid
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Transformation of Plant Cells by the Ti Plasmid
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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

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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
  T-DNA (transferred DNA)
• The T-DNA resides on a large plasmid called the
  Ti (tumor inducing) plasmid found in A.
  tumefaciens

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The Ti plasmid of Agrobacterium tumafaciens and
the transfer of its T-DNA to the plant nuclear
genome
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Ti plasmid structure function

• 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.

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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
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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
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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.

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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
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Genetically Modified Organisms