Difference between revisions of "Part:BBa K518000"

Revision as of 13:01, 25 September 2011

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.>

Molecular Biology

Firefly luciferase is a 62 000 Dalton monomeric protein. The enzyme catalyzes ATP-dependent D-luciferin oxidation with light emission centered on 560 nm. With excessive amount of substrate, its luminescence is directly proportional to the number of the enzyme molecules.

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