IGEM:IMPERIAL/2007/Projects/Reporters

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

• Gene name: gfp
• Gene sequence: 5170 bp, GenBank
• Part: BBa_E0040
• Description:
• Response Time: ~1 hour
• Steady State Time: 8 hours
• Concentration sensitivity: High
• Advantages
• Disadvantages
  • Slow folding

Red Fluorescent Protein

• Gene name: rfp
• Part: ???
• Description:
• Response Time: ~8 hours
• Steady State Time: 12++ hours
• Concentration sensitivity: ???
• Advantages
• Disadvantages