IGEM:IMPERIAL/2007/Projects/Reporters

Useful Reporters

I have classified reporters into three main classes for the sake of simplicity.

Bioluminescence

These reporters code for an enzyme which cleaves an added substrate, giving off light which is observed as luminescence. 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

There reporters code for a fluorescent molecule, which has to be activated by light of a certain wavelength, allowing them to emit light of a different wavelength. Advantages:

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

Disadvantages:

• Slow folding time, resulting in generally slow response
• Can require the use of UV light

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

These reporters code for a specific enzyme, which can metabolize an added substrate to give a chromophore. Advantages:

• Usually colourmetric and visible under light
• Generally fast expression times
• Assay after expression allows for much higher concentration sensitivity

Disadvantages:

• Requires the addition of extra substrates
• Enzyme concentration is generally assayed after expression (sample is taken from cell culture and assayed)
• Enzymes are generally already found in many strains of E. coli

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:
  • Visible under white light
• 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