Difference between revisions of "Part:BBa K1993018"
 
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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.  
 
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.  
 
We had constructed two kinds of luciferases. One of them was firely luciferase that is from Photinus pyralis[1].
 
We had constructed two kinds of luciferases. One of them was firely luciferase that is from Photinus pyralis[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].
+
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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The chemical reaction catalyzed by firefly luciferase takes place in two steps:<br>
 
The chemical reaction catalyzed by firefly luciferase takes place in two steps:<br>
  
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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 purified the gene Luciferase(Figure 2), we modified our mesenchymal stem cells(MSCs) by adding such gene under the control of EF-1a, then tested inside a mouse
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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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'''Figure 2 purification of gene Luciferase '''
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'''Figure 2 Purification of Luciferase '''
  
 
==References==
 
==References==

Latest revision as of 19:20, 17 October 2016


Luciferase(Firefly Luciferase)


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. We had constructed two kinds of luciferases. One of them was firely luciferase that is from Photinus pyralis[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].

The chemical reaction catalyzed by firefly luciferase takes place in two steps:

• luciferin + ATP → luciferyl adenylate + PPi
• luciferyl adenylate + O2 → oxyluciferin + AMP + light
Light is produced because the reaction forms oxyluciferin in an electronically excited state. The reaction releases a photon of light as oxyluciferin goes back to the ground state.

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] Gould SJ, Subramani S (Nov 1988). "Firefly luciferase as a tool in molecular and cell biology". Analytical Biochemistry. 175 (1): 5–13. 

[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] Baldwin TO (Mar 1996). "Firefly luciferase: the structure is known, but the mystery remains". Structure. 4 (3): 223–8

[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
    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 808