Difference between revisions of "Part:BBa K518002"
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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). | 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). | + | 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). Again, we can easily get the numerical information of how many molecules are translated, not just the arbitrary luminescence units. |
[[Image:Calibration1.png|500px]] | [[Image:Calibration1.png|500px]] | ||
Revision as of 04:00, 4 October 2011
Firefly-renilla dual luciferase assay kit
Luciferase assay is one of the most popular reporter assay system for quantitatively measuring the function of promoters and other cis-elements. The wide-ranging and quantitative detection is the prominent feature of this assay; light emmision from 10~(-18) ― 10~(-11) mol protein can be quantitatively measured using luminometer (see our results). A promoter or other cis-element to be analysed can be ligated above this part. Its effect will be measured as an altered firefly luciferase expression. Meanwhile, renilla luciferase expression can be used to as an internal control.
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). Again, we can easily get the numerical information of how many molecules are translated, not just the arbitrary luminescence units.
<Fig 1. Estimation of firefly luciferase amount by linear regression.>
Usage
We performed a test of this cassette using BBa_J23119 and BBa_R0011. We successfully verified the IPTG-dependence of BBa_R0011, and at the same time, the potency of this part as an evaluation tool. For experimental and analytical details, see [http://2011.igem.org/Team:UT-Tokyo our page].
<Fig.1: Time-dependent luminescence measurement of firefly luciferase. The first graph shows the raw data obtained by triplicate experiments. Data is presented as mean ± S.D.. The second graph is a model fitting according to the enzymatic kinetics theory.>
<Fig.2: Time-dependent luminescence measurement of renilla luciferase.>
<Fig.3: Demonstration of promoter evaluation. Relative Promoter Unit (RPU) is calculated as: (maximum of firefly luminescence) / (maximum of renilla luminescence). Data is obtained from triplicate experiments. Data is expressed as mean±S.D..>
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1790
Illegal NheI site found at 1813 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2392
Illegal SapI.rc site found at 827