IGEM:IMPERIAL/2007/Projects/Reporters

Useful Reporters

Bioluminescence

Advantages:

• More sensitive than other spectroscopy reporters, since there is often neglectable background bioluminescence (not naturally found in cells).
• 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
• Gene sequence: 1781 bp, GenBank
• 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?

Renilla luciferase

• Gene name: luci
• Part: BBa_J52008
• Description: Requires renilla luciferin, which is oxidized by oxygen to genrate light. The only luciferase part in the registry.
• Enzyme: EC 1.13.12.5
• Reaction: Renilla luciferin + O₂ <=> oxidized Renilla luciferin + CO₂ + light
• Response Time: ???
• Steady State Time: ???
• Concentration sensitivity: Moderate
• Advantages:
• Disadvantages:
  • Substrate limiting

Fluorescence Reporters

Advantages:

• They are highly stable, and can be active even in dead cells
• Does not require additional substrates, only light excitation
• Can be fused with other proteins

Disadvantages:

• Slow folding time, resulting in generally slow response

Green Fluorescent Protein

• Gene name: gfp
• Gene sequence: 5170 bp, GenBank
• Part: BBa_E0040
• Description: protein which can be exicited under 488 nm, releasing emission at 509 nm that is detected as a green light.
• Response Time: ~1 hour
• Steady State Time: 8 hours
• Concentration sensitivity: High
• Advantages
  • GFP can be fused with other proteins
  • Highly stable, can be visible in dead cells
• Disadvantages
  • Requires UV excitation
  • Slow folding

Red Fluorescent Protein

• Gene name: rfp
• Part: BBa_E1010
• Description: Fluorescent protein which can be excited under 584 nm, releasing emission at 607 nm that is detected as a red light.
• Response Time: ~8 hours
• Steady State Time: 12++ hours
• Concentration sensitivity: Moderate (due to long folding hours)
• Advantages:
  • Visible under normal light
• Disadvantages:
  • Very slow folding

Enzymic Reporters

Advantages:

• Usually colourmetric and does not require sophisicated equipment

Disadvantages:

• Requires killing of the cell to release enzymes
• Substrate specific

Alkaline Phosphatase

• Gene name: ppb
• Part: BBa_J61032
• Description: Enzyme which functions optimally in alkaline environments (periplasm), breaks down para-nitrophenol phosphate to form compound which absorbs at 405 nm. Sometimes known as SEAP (secreted akaline phosphatase).
• Enzyme: EC 3.1.3.1
• Reaction: Phosphate monoester + H₂O <=> an alcohol + phosphate
• Response Time: ??? (possibly within minutes)
• Steady State Time: ???
• Concentration sensitivity: High
• Advantages:
  • High concentration sensitivity
• Disadvantages:
  • Enzymatic reaction is slow

β-D-Galactosidase

• Gene name: lacZ
• Part: BBa_E0033 Note: This part is special and works only when both fragments of lacZ are present.
• Description: β-galactosidase cleaves a synthetic lactose homologue, o-nitrophenyl-β-D-galactoside (ONPG) to give a yellow compound, o-nitrophenol, which has a peak absorbance at 420 nm. Using Miller's equation, the amount of β-galactosidase can be determined.
  Alternatively, 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal) or variants (such as S-gal) can be also be cleaved into a coloured compound absorbing at 420 nm.
• Enzyme: EC 3.2.1.23
• Reaction: o-nitrophenyl-β-D-galactoside + H₂O <=> galactose + o-nitrophenol
• Response Time: Within minutes
• Steady State Time: 2-3 hours
• Concentration sensitivity: Very high (with Miller)
• Advantages:
  • Visible under white light, good for selection purposes
  • Very high concentration sensitivity
  • Enzyme reaction can be stopped during assay
• Disadvantages:
  • Must use lacZ^- strain of E. coli to prevent cross-talking
  • Requires cells to be killed to release enzymes for Miller's assay

β-D-Glucuronidase

• Gene name: gus
• Description: Acts similarly to β-galactosidase, can hydrolyze 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) into a coloured compound, which absorbs at ??? nm. Alternatively, 4-Methylumbelliferyl-β-D-glucuronide (MUG) can be hydrolyzed into a fluorescent compound, which absorbs at 390 nm and emits at 480 nm.
• Enzyme: EC 3.2.1.31
• Reaction: A beta-D-glucuronoside + H₂O <=> D-glucuronate + an alcohol
• Response Time: ??? (possibly within minutes)
• Steady State Time: ???
• Concentration sensitivity: ???
• Advantages:
• Disadvantages:
  • β-glucuronidase naturally in E. coli might interfere with expression of recombinant gene

Other Possible Reporters

• Chloramphenicol Acetyltransferase (CAT)
• Human Growth Hormone (hGH)

Detecting Reporters

• Spectrometric Assays
• Enzyme Activity Assays
• Immunology Assays