Determination of Nitrate Flux in Transgenic Tobacco Plants

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Determination of Nitrate Flux in Transgenic
Tobacco Plants

• Presentation by
• Tasha Witham and
• Violetta Priestley

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Problems With Nitrogen Pollution
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Nitrates as a Pollutant
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Goals of Dr. Knights Transgenic Research

• To increase nitrogen use efficiency in plants.

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Transgenic MethodsHow are these plants
transformed?

• Crown Gall Disease in Plants

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Agrobacterium tumefaciens

• A. tumefaciens induces gall formation
• The Ti plasmid of A. tumefaciens has 3 genes

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Creation of a Transgenic Plant

• Modifying Ti plasmids
• Loading of Modified Ti plasmids
• Transference of Modified Ti plasmids
• Formation of Transgenic plants

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Goal of Our Chemical Research

• To measure nitrate flux in transgenic tobacco
  plants
• Determination of nitrate flux
• -Direct determination with nitrate measurement
• -Indirect determination by measuring the enzyme
  and protein levels

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Our Chemical Research Method

• Created two standard absorption curves, an enzyme
  standard curve gammaglutamylhistamine synthetase
  (GHA) and a protein standard curve using bovine
  serum albumin (BSA)

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Method for Creating the Enzyme Standard Curve

• (1) Create a stock solution of GHA in distilled
  water 1mg/1ml
• (2) Graded dilution with dH2O 1 part GHA stock
  to 9 parts dH2O, 2 parts GHA to 8 parts dH2O,
  etc.
• (3) Addition of 3 ml FeCl2/FCA/HCL after 15
  minutes

• (4) Molarity calculations based on GHA molar mass
  of 146 g/mol. Calculation (0.100mg x
  0.001)/146g)/3.100 ml
• (5) Measure absorbance of each graded solution
  using an ICP spectrophotometer at 540 nm
• (6) Create a standard curve of absorbance vs.
  GHA

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Method for Creating The Protein Standard Curve

• (1) Create a stock solution of BSA in water
  1mg/1ml
• (2) Graded dilution with dH2O 1 part BSA to 9
  parts dH2O, 2 parts BSA to 8 parts dH2O, etc.
• (3) Addition of 5 ml working Bradfords solution
  (1 part stock Bradfords to 2.5 parts dH2O)
• (4) Measure absorbance of each graded solution
  with spectrophotometer at 595nm
• (5) Standard curve of absorbance vs. BSA

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Enzyme Assay Method

• (1) Initial weighted sample of in 3ml of buffer
  solution (TOB IM buffer)
• (2) Extraction of 0.100 ml of root and 0.050 ml
  of leaf plus 0.500ml of assay mix

• (3) Addition of 2ml FeCl2/FCA/HCL after 15
  minutes
• (4) Molarity calculation based on GHA molar mass
  of 146 g/mol

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Protein Assay Method

• (1) Initial weighed sample in 3ml of buffer
  solution (TOB IM buffer)
• (2) Extraction of 0.100ml
• (3) Addition of 5ml working Bradfords

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Leaf/Root Ratios

• Wild Type enzyme potential3.980
• PN-18 enzyme potential29.122
• PN-99 enzyme potential 15.654
• Wild Type protein1.443
• PN-18 protein1.495
• PN-99 protein2.161