LUCIFERASE AND ITS APPLICATIONS

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LUCIFERASE AND ITS APPLICATIONS

• KARTHIKEYAN NARAYANAN

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Bioluminescence !!!

• Bioluminescence, (living light) is ability of
  living organisms to produce light through a
  chemical reaction.
• There are two ways an organism can
  bioluminescence

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What is fluorescence ?
In fluorescence, energy from a source of light is
absorbed and reemitted as another photon. In
bioluminescence the excitation energy is supplied
by a chemical reaction rather than from a source
of light.
Fluorescence mechanism
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Why do they produce light ?

• To find food

To find mates
For Defense against predators
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What produces light?
Two chemicals are required. Luciferin which
produces the light generically Luciferase that
drives or catalyzes the reaction
D.Smith
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Types of Luciferin
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Types of Luciferase
Bacterial luciferase showing alpha and beta
peptides.
Firefly luciferase with ATP (shown magenta)
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Types of Luciferases
Coelenterate luciferase Also called as Aequorin
The balls (yellow) shows the bound Ca in the
enzyme The light is the reaction of the Ca with
the aequorin
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LUCIFERASE GENES
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Bacterial Luciferase gene (Vibrio fischeri)

• lux I- autoinducer synthesis,
• lux R- receptor molecule that binds to the
  autoinducer, controls the transcription of the
  right operon
• lux C, D and E for aldehyde production.
• lux A and B for a and ß peptides.

Meighen, 1991
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Autoinduction
V.fischeri cells exhibit autoinduction in the
organs of their host
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Bacterial Luciferase action
Relationship between the bacterial
bioluminescence reaction and the genes and
enzymes linked to the lux operon
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Dinoflagellate luciferase gene

• Gonyaulax polyedra luciferase gene.
• This system has two types of proteins luciferin
  binding protein and luciferase
• The gene has three tandem repeats
• The gene is transcribed only once a day in a
  circadian rhythm fashion

Lee et al., and Bae and Hastings, 1993 and 1994
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Firefly luciferase gene

• Luciferase gene was cloned in E.coli by Wet et
  al., in 1985
• It was then successfully cloned in tobacco
  plants.

Hotaria grp luciferase gene (Choi et al., 2003)
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APPLICATIONS
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Applications

• Animal Sciences
• Environmental sciences
• Plant sciences
• Entomology

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Animal sciences

• Imaging cells In vivo
• Two different luciferases along with different
  substrates for each and their kinetics can be
  studied in vivo in the tissue

Bioluminescence from C6-Fluc cells (A) shows a
relatively strong signal beyond 10 min (Ghambir
2002)
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Animal sciences

• In Gene therapies
• Luminescent markers play a critical role in gene
  therapy to achieve controlled and effective
  delivery of genes to target cells avoiding
  ectopic expression

In vivo transcriptional assay in lung after
transfection of pBI-L/heme oxygenase (HO)-1. Weng
et al., 2000
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Animal science

• Transgenic animal research

Mouse model for retinoblastoma suppressor
gene-dependent pituitary cancer development with
co-expression of fluc genes enabling long term
bioluminescence imaging, quantification of tumour
and assessment of chemotherapeutic response
Transgenic mice that express the fluc gene under
the control of the nuclear factor ?B (NF-?B)
promoter. real-time imaging of the NF-?B promoter
activity
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Environmental Science

• Luminous systems are used as biosensors to
  monitor environmental toxicity.
• Fishes are transformed with luciferases to
  monitor the level of tetracycline in their
  environment
• Some bacteria are transformed with luciferases to
  monitor the toxic gases in mines

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Plant Science

• Tobacco plants are cloned with luciferase gene to
  express the enzyme and glow when dipped in
  luciferin. (Ow et al., 1986)

Luciferases are used to monitor the circadian
rhythms in plants
Transformed Arabidopsis plants with Phytochrome A
(phy A) gene promoter fused with luciferase
reporter to study the rhythms in plants
associated with the gene.
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Plant Science

• To study the stress response in plants

Luciferin when sprayed into an adult plant
responds very late than a plant that is wounded.
This demonstrates that there are some barriers
that restrict the luciferin entry in adult
plants, which may cause unavailability of
luciferin and a limiting factor for non-invasive
luciferase assays
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Plant Science
Finding genes faster

• chimeric agrobacterium-luciferase genes are
  transformed into the plant to induce mutations in
  the genes and make them nonfunctional. the
  consequences of that plants response to the
  environment is observed. screening the plants
  that are react abnormally, are chosen and
  separated from normal plants.

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Entomology

• To develop ways to combat diseases spread by
  vectors
• To develop strategies to tackle insect problems
  in crops
• Silk worms that produce fluorescent green fibres
  have been created by Japanese genetic engineers,
  opening up new opportunities for the fabric
  industry.

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Conclusion

• The luciferase systems clearly reflect the
  powerful and possibly transformative
  technologies.
• The potential power of this research helps to see
  the fundamental biological processes in a new
  light
• It will not only help to enhance our knowledge
  and understanding, but also accelerate the rate
  of discovery in the field of biological sciences

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WISHING U A GLOWING CHRISTMAS AND A HAPPY NEW
YEAR 2004
A genetically engineered Christmas tree
containing jellyfish luciferase that glows at
night developed in UK.