IGEM:IMPERIAL/2007/Projects/Reporters: Difference between revisions

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**Substrate limiting
**Substrate limiting
Questions: What substrate does this need?
Questions: What substrate does this need?
===Fluorescence Reporters===
'''Advantages''':
*
'''Disadvantages''':
*Slow folding time, resulting in generally slow response
====Green Fluorescent Protein====
*Gene name: ''gfp''
*Part: [http://parts.mit.edu/registry/index.php/Part:BBa_E0040 BBa_E0040]
*'''Description''':
*

Revision as of 07:10, 27 July 2007

Useful Reporters

GUS reporter system

  • Description: Analyze the activity of a promoter (in terms of expression of a gene under that promoter) either in a quantitative way or through visualization of its activity in different tissues. The technique is based on beta-glucuronidase, an enzyme from the bacterium Escherichia coli. This enzyme when incubated with some specific colorless or not fluorescent substrates, can transform them into coloured or fluorescent products.
  • Potential use: Fluorescent output
  • Link: http://en.wikipedia.org/wiki/GUS_reporter_system

Bioluminescence

Advantages:

  • More sensitive than other spectroscopy reporters, since there is often neglectable background bioluminescence.
  • Relatively fast response

Disadvantages:

  • Generally metabolically stressing, since key metabolic compounds are required in the generation of light.
  • Usually requires the addition of substrates to be oxidized, which can limit the luminescence signal.

Firefly luciferase

  • Gene name: lucff
  • Description: Oxidizes luciferin with the help of oxygen, releases light as a product of oxidation.
  • Enzyme: EC 1.13.12.7
  • Reaction: Photinus luciferin + O2 + ATP <=> oxidized Photinus luciferin + CO2 + AMP + diphosphate + light
  • Response Time: ~30 minutes
  • Steady State Time: 4 hours
  • Concentration sensitivity: Moderate
  • Advantages:
    • Fast response
    • Can be initiated by addition of luciferin
  • Disadvantages:
    • Metabolic stress, ATP is consumed
    • Substrate limiting

Bacterial luciferase

  • Gene name: luxCDABE
  • Description: 5 subunit protein which oxidizes FMN with the help of oxygen to generate FMN and H2 complex. This complex breaks down to form light.
  • Enzyme: EC 1.14.14.3
  • Reaction: RCHO + reduced FMN + O2 <=> RCOOH + FMN + H2O + light
  • Response Time: ~30 minutes
  • Steady State Time: 8 hours
  • Concentration sensitivity: Moderate
  • Advantages:
    • Fast response
    • Can be initiated by addition of substrate
  • Disadvantages:
    • Five subunits required
    • Metabolic stress, FMN is consumed
    • Substrate limiting

Questions: What substrate does this need?

Fluorescence Reporters

Advantages:

Disadvantages:

  • Slow folding time, resulting in generally slow response

Green Fluorescent Protein

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