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Lecture 5. Reporter genes. Reporter Genes . A gene encoding an enzyme medium modification is added along with your gene nucleic acid sequences encoding easily assayed proteins

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lecture 5

Lecture 5

Reporter genes

reporter genes
Reporter Genes
  • A gene encoding an enzyme medium modification is added along with your gene
  • nucleic acid sequences encoding easily assayed proteins
  • Reporter genes include -galactosidase (encoded by lacZ), -glucuronidase (encoded by uidA), chloramphenicol acetyltransferase, luciferase and green fluorescent protein (GFP) .
novel reporter genes
Novel Reporter Genes
  • Luciferase - gene from fireflies with causes the glow - add a substrate to tissue that has been transformed and it lights up
  • Green Fluorescent Protein - from jellyfish - under lights and filter the transgenic plants will fluoresce
  • GUS - glucuronidase gene will convert added substrate to blue color.
glucuronidase gus
ß-glucuronidase (GUS)
  • GUS is probably the most widely used reporter gene in plants
  • low endogenous activity in plant
  • stable enzyme which hydrolyses wide range of ß-glucuronides
  • easily assayed for histochemical analysis, using X-gluc (5-bromo, 4-chloro, 3-indoyl ß–glucuronide).
  • After cleavage, oxidation of the indole derivative causes dimerisation and the production of an insoluble indigo dye
gus expression
GUS expression
  • The GUS gene creates blue coloration of transformed tissue when transformed cells or tissues are provided with the appropriate substrate.
luciferase luc
Luciferase (Luc)
  • enzyme from firefly (Photinus pyralis)
  • produces flashes of light in the presence of luciferin and ATP
  • detected in tissue extracts or even in the intact plant after watering with luciferin
  • allows non-destructive imaging of plants
green fluorescent protein gfp
Green fluorescent protein (GFP)
  • from the jelly-fish Aequoria victoria
  • intrinsically fluorescent
  • due to a chromophore in the protein by cyclisation and oxidation of the amino acids Ser-Tyr-Gly at positions 65-67 in the polypeptide
  • allows non-destructive imaging of plants and sub cellular localization of GFP by microscopy
  • several variants of GFP to give different colours – YFP (yellow), BFP (blue), CFP (cyan)
  • produced by alteration in the chromophore (Tyr66), or residues close to the chromophore in the 3-D protein structure
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