Difference between revisions of "Part:BBa K518000"
 
(11 intermediate revisions by 2 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K518000 short</partinfo>
 
<partinfo>BBa_K518000 short</partinfo>
  
−
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.
+
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===
 
===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).
+
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).
  
−
[[Image:Calibration1.tif]]
+
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:Calibration2.tif]]
+
[[Image:Calibration1.png|500px]]
 +
 
 +
<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===
 
===Usage===
−
For detailed results of this cassette, see [http://2011.igem.org/Team:UT-Tokyo our page] and [[Part:BBa_K518002 |BBa_K518002]].
+
For detailed data obtained, see [http://2011.igem.org/Team:UT-Tokyo our page] and [[Part:BBa_K518002 |BBa_K518002]].
  
 
===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).
 +
 
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).
 +
 
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)
  
Line 26: Line 33:
  
  
−
<!-- Uncomment this to enable Functional Parameter display
+
<!-- -->
 +
<!--
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K518000 parameters</partinfo>
 
<partinfo>BBa_K518000 parameters</partinfo>
 
<!-- -->
 
<!-- -->

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).

Calibration1.png

<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


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]
    Illegal SapI.rc site found at 827