Part:BBa_K518000

RBS + firefly luciferase + d.terminator

Luciferase from firyfly (Photinus pyralis) expression cassette. Firefly luciferase emits light by the oxidation of D-luciferin, its substrate. Luciferase can be used as a measuring tool when combined with other cis-elements (regions regulating the expression of genes on the same DNA molecule; promoters and other protein-binding domains), because of its highly quantitative performance.

Calibration

We performed a calibration for Photynus pyralis (Firefly) luciferase using standard reagents of enzyme and D-luciferin. Reagents were granted from Berthold Japan (Tokyo, Japan). Luminescence was measured using a luminometer GENE LIGHT 200 from MICROTEC, Co., Ltd. (Chiba, Japan).

The result indicates that under 1000 luciferase molecules can be detected, and that luciferase amounts can be well estimated by a simple linear regression in a logarithmically seven-digit range (from 10^(-20) to 10^(-13) mol).

<Fig 1. Linear regression.>

<Fig 2. Sigmoid regression.>

Usage

For detailed data obtained, see [http://2011.igem.org/Team:UT-Tokyo our page] and BBa_K518002.

Reference

Bronstein I., et.al., Chemiluminescent and Bioluminescent reporter gene assays. Anal. Biochem, 219, 169 (1994). DeLuca M., et al., Firefly Luciferase: Mechanism of action, cloning and expression of the active enzyme. J. Biolum. Chemilum. 3, 1 (1989). Gould, S.J. and S. Subramani, Firefly luciferase as a tool in molecular and cell biology. Anal. Biochem. 175:5-13 (1988)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

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