Difference between revisions of "Part:BBa K518000"
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| − | Luciferase from | + | Luciferase from firefly (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. |
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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). | 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). | ||
| − | [[Image:Calibration1. | + | [[Image:Calibration1.png|500px]] |
| − | < | + | <Figure. Estimation of firefly luciferase amount by linear regression.> |
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===Molecular Biology=== | ===Molecular Biology=== | ||
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===Reference=== | ===Reference=== | ||
Bronstein I., et.al., Chemiluminescent and Bioluminescent reporter gene assays. Anal. Biochem, 219, 169 (1994). | Bronstein I., et.al., Chemiluminescent and Bioluminescent reporter gene assays. Anal. Biochem, 219, 169 (1994). | ||
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DeLuca M., et al., Firefly Luciferase: Mechanism of action, cloning and expression of the active enzyme. J. Biolum. Chemilum. 3, 1 | DeLuca M., et al., Firefly Luciferase: Mechanism of action, cloning and expression of the active enzyme. J. Biolum. Chemilum. 3, 1 | ||
(1989). | (1989). | ||
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Gould, S.J. and S. Subramani, Firefly luciferase as a tool in molecular and cell biology. Anal. Biochem. 175:5-13 (1988) | Gould, S.J. and S. Subramani, Firefly luciferase as a tool in molecular and cell biology. Anal. Biochem. 175:5-13 (1988) | ||
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===Functional Parameters=== | ===Functional Parameters=== | ||
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<partinfo>BBa_K518000 parameters</partinfo> | <partinfo>BBa_K518000 parameters</partinfo> | ||
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Latest revision as of 23:49, 3 October 2011
RBS + firefly luciferase + d.terminator
Luciferase from firefly (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).
<Figure. Estimation of firefly luciferase amount by linear 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 827