Difference between revisions of "Part:BBa J52008"
 
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===Usage and Biology===
 
===Usage and Biology===
 
This part contains a Eukaryotic ribosome binding site and non-standard prefix and suffix.  See the sequence first.   
 
This part contains a Eukaryotic ribosome binding site and non-standard prefix and suffix.  See the sequence first.   
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<head></head>
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<h3>This part was characterized by the iGEM team DUT_China_B in 2019.</h3><br><br>
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<h1>Usage</h1><br>
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Renilla-luciferin 2-monooxygenase,also known as Renilla luciferase, or RLuc, is a bioluminescentenzyme found in Renilla reniformis, belonging to a group of coelenterazine luciferases. Of this group of enzymes, the luciferase from Renilla reniformis has been the most extensively studied, and due to its bioluminescence requiring only molecular oxygen, has a wide range of applications, with uses as a reporter gene probe in cell culture, in vivo imaging, and various other areas of biological researches[1]. <br>
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RLuc catalyzes the chemical reaction ：<br>
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Coelenterazine + O2→coelenteramide + CO2 + hν <br>
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In the process, coelenterazine is oxidized with a concurrent loss of CO2, and a photon of blue light is emitted[2].<br><br>
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<h1>Characterization</h1><br>
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For the configuration, preservation and use of coelenterin solution,<a href="https://2019.igem.org/wiki/images/3/30/T--DUT_China_B--4_protocols%EF%BC%9AAbsorption_measurement_of_Fluorescence.pdf"> see protocol Absorption measurement of Fluorescence.pdf</a><br>
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Determination of optimal incubation time of Rluc. <br>
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After the 3 mL system is configured, 2900 uL crude enzyme solution and 100 uL coelenterin working solution should be added, and  then measure the luminescence intensity at 480 nm after incubation for 0,5,10,15,20,25,30 min.  <br>
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Determination of optimal substrate concentration of Rluc.<br>
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The system was configured to 3 mL, and an appropriate amount of crude enzyme solution and the prepared coelenterin working solution should be added to make its working concentration 0,5,10,15,20 uM respectively. After incubation for 10 min, measure the luminescence intensity at 480 nm .<br><br>
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<h1>Results and Discussion</h1><br>
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As can be seen from the Figure 1 and Table 1, the relative luminescence intensity increased with the increase of incubation time, and the growth rate was faster in the first 10 min.<br>
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<img src="https://2019.igem.org/wiki/images/4/40/T--DUT_China_B--rluc1%E8%A1%A8%E6%A0%BC1.png">
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<img src="https://2019.igem.org/wiki/images/f/fc/T--DUT_China_B--rluc_figure_1.png"><br>
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As can be seen from the Figure 2 and Table 2, the maximum luminescence intensity appeared at the substrate of 10 uM, so the subsequent experimental coelenterin substrate concentration was determined to be 10 uM.<br>
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<img src="https://2019.igem.org/wiki/images/6/69/T--DUT_China_B--rluc2%E8%A1%A8%E6%A0%BC2.png">
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<img src="https://2019.igem.org/wiki/images/4/41/T--DUT_China_B--rluc_figure_2.png">
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<h1>references</h1>
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[1]Daunert S, Deo S K. Photoproteins in Bioanalysis[J]. Wiley, 2006.
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[2]Schomburg D, Salzmann M. Enzyme Handbook[J]. 2001.
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<partinfo>BBa_J52008 parameters</partinfo>
 
<partinfo>BBa_J52008 parameters</partinfo>
 
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===experiment===
 
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We transferred the plasmid with only carrying hRluc and stimulated them with different concentration of sugar. With the purpose of detecting the correlation between the expression of hRluc and the stimulation of the sugar’s concentration, we tracked the cells and used the double luciferase kit to test their correlation. And the result showed below.
 
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[[File:T--NUDT CHINA--result of SV40 promoter.png|130px|]]
 

Latest revision as of 00:27, 22 October 2019

luciferase: luciferin 2-monooxygenase from Renilla reniformis (EC 1.13.12.5; SwissProt:: P27652)

Rluc is a protein called Renilla luciferase and it emits light when the right substrate is added. That is why it can be used as a reporter to track other proteins or to monitor the activity of a promoter.

Usage and Biology

This part contains a Eukaryotic ribosome binding site and non-standard prefix and suffix. See the sequence first.

This part was characterized by the iGEM team DUT_China_B in 2019.



Usage


Renilla-luciferin 2-monooxygenase,also known as Renilla luciferase, or RLuc, is a bioluminescentenzyme found in Renilla reniformis, belonging to a group of coelenterazine luciferases. Of this group of enzymes, the luciferase from Renilla reniformis has been the most extensively studied, and due to its bioluminescence requiring only molecular oxygen, has a wide range of applications, with uses as a reporter gene probe in cell culture, in vivo imaging, and various other areas of biological researches[1].
RLuc catalyzes the chemical reaction ：
Coelenterazine + O2→coelenteramide + CO2 + hν
In the process, coelenterazine is oxidized with a concurrent loss of CO2, and a photon of blue light is emitted[2].

Characterization


For the configuration, preservation and use of coelenterin solution, see protocol Absorption measurement of Fluorescence.pdf
Determination of optimal incubation time of Rluc.
After the 3 mL system is configured, 2900 uL crude enzyme solution and 100 uL coelenterin working solution should be added, and then measure the luminescence intensity at 480 nm after incubation for 0,5,10,15,20,25,30 min.
Determination of optimal substrate concentration of Rluc.
The system was configured to 3 mL, and an appropriate amount of crude enzyme solution and the prepared coelenterin working solution should be added to make its working concentration 0,5,10,15,20 uM respectively. After incubation for 10 min, measure the luminescence intensity at 480 nm .

Results and Discussion


As can be seen from the Figure 1 and Table 1, the relative luminescence intensity increased with the increase of incubation time, and the growth rate was faster in the first 10 min.

As can be seen from the Figure 2 and Table 2, the maximum luminescence intensity appeared at the substrate of 10 uM, so the subsequent experimental coelenterin substrate concentration was determined to be 10 uM.

references

[1]Daunert S, Deo S K. Photoproteins in Bioanalysis[J]. Wiley, 2006. [2]Schomburg D, Salzmann M. Enzyme Handbook[J]. 2001.

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 BsaI.rc site found at 547