Difference between revisions of "Part:BBa K1993015"
 
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Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is distinct from a photoprotein. A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. So, multiple luciferases had been found and used in biological researches.  
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Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is distinct from a photoprotein. A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. Hence, multiple luciferases had been found and used in biological researches.  
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We had constructed two kinds of luciferases. One of them was Renilla-luciferin 2-monooxygenasethat is from Renilla reniformis [1].
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We had constructed two kinds of luciferases. One of them was Renilla-luciferin 2-monooxygenase that is from Renilla reniformis [1].
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Unlike other fluorescent proteins, light excitation is not needed for luciferase bioluminescence, which means minimal autofluorescence and therefore virtually background-free fluorescence[2]. In other words, since luciferase reaction is a enzymatic reaction, light is emitted when luciferase acts on the appropriate luciferin substrate. Photon emission can be detected by light sensitive apparatus such as aluminometer or modified optical microscopes. This allows observation of biological processes[3].
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Unlike other fluorescent proteins, light excitation is not needed for luciferase bioluminescence, which means minimal autofluorescence and therefore virtually background-free fluorescence[2]. In other words, since luciferase reaction is an enzymatic one, light is emitted when luciferase acts on the appropriate luciferin substrate. Photon emission can be detected by light sensitive apparatus such as autoluminometer or modified optical microscopes. This allows observation of biological processes[3].
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RLuc catalyzes the chemical reaction
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RLuc catalyzes the chemical reaction:
  
 
Coelenterazine + O2 → coelenteramide + CO2 + hν
 
Coelenterazine + O2 → coelenteramide + CO2 + hν
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'''Figure 1 Mechanism for Luciferase [4].'''
  
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'''Figure 1 Mechanism for luciferase [4].'''
 
  
 
In biological research, luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest[5]. But in our project, we used luciferase for another purpose: trace the cells non-invasively inside a live mouse using a sensitive charge-couple device camera.  
 
In biological research, luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest[5]. But in our project, we used luciferase for another purpose: trace the cells non-invasively inside a live mouse using a sensitive charge-couple device camera.  
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Once we constructed the luciferase, we modified our Mesenchymal stem cells(MSCs) by adding such plasmid, then tested inside a mouse(Figure 2). From the picture, we can clearly know the precise location of the MSCs in vivo.  
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After we purified the gene luciferase(Figure 2), we constructed a plasmid that contained constitutive promoter EF-1α and it.
  
 
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<img src="https://static.igem.org/mediawiki/2016/6/6c/T--SYSU-MEDICINE--luciferase1-2.png" style="width:200px"  ></a>
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<img src="https://static.igem.org/mediawiki/2016/1/11/T--SYSU-MEDICINE--Luciferase1.jpeg" style="width:200px"  ></a>
  
 
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'''Figure 2. Luciferase indicating the position of MSCs in vivo'''
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'''Figure 2 Purification of Luciferase'''
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==References==
 
==References==

Latest revision as of 19:21, 17 October 2016


Luciferase (Renilla reniformis)

Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is distinct from a photoprotein. A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. Hence, multiple luciferases had been found and used in biological researches. We had constructed two kinds of luciferases. One of them was Renilla-luciferin 2-monooxygenase that is from Renilla reniformis [1]. Unlike other fluorescent proteins, light excitation is not needed for luciferase bioluminescence, which means minimal autofluorescence and therefore virtually background-free fluorescence[2]. In other words, since luciferase reaction is an enzymatic one, light is emitted when luciferase acts on the appropriate luciferin substrate. Photon emission can be detected by light sensitive apparatus such as autoluminometer or modified optical microscopes. This allows observation of biological processes[3].

RLuc catalyzes the chemical reaction:

Coelenterazine + O2 → coelenteramide + CO2 + hν

In the process, RLuc is oxidized, and a photon of blue light is emitted.

Figure 1 Mechanism for Luciferase [4].


In biological research, luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest[5]. But in our project, we used luciferase for another purpose: trace the cells non-invasively inside a live mouse using a sensitive charge-couple device camera.

After we purified the gene luciferase(Figure 2), we constructed a plasmid that contained constitutive promoter EF-1α and it.

Figure 2 Purification of Luciferase


References

[1] Loening, A. M.; Fenn, T. D.; Gambhir, S. S. (2007). "Crystal Structures of the Luciferase and Green Fluorescent Protein from Renilla reniformis". Journal of Molecular Biology. 374 (4): 1017–1028

[2] Williams TM, Burlein JE, Ogden S, Kricka LJ, Kant JA (Jan 1989). "Advantages of firefly luciferase as a reporter gene: application to the interleukin-2 gene promoter".Analytical Biochemistry. 176 (1): 28–32.

[3] "Introduction to Bioluminescence Assays". Promega Corporation. Retrieved2009-03-07.

[4]  Anderson, JM; Charbonneau, H; Cormier, MJ (12 March 1974). "Mechanism of calcium induction of Renilla bioluminescence. Involvement of a calcium-triggered luciferin binding protein.".Biochemistry. 13 (6): 1195–200

[5]Fan F, Wood KV (Feb 2007). "Bioluminescent assays for high-throughput screening". ASSAY and Drug Development Technologies. 5 (1): 127–36.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 228
    Illegal BamHI site found at 771
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]