Difference between revisions of "Part:BBa K2440008"
(→‎Usage and Biology)
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===Usage and Biology===
 
===Usage and Biology===
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Previously, iGEM07_Ljubljana had already designed the part BBa_I712019, containing the coding sequence of Firefly Luciferase which had fulfilled function of observation in vivo.
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This year,this part was improved by us to optimize its function in a dual-luciferase miRNA target expression system. Two restriction sites of BsmBI were added to the 3’ end of firefly luciferase, through which, the 3’ untranslated region of any gene can be inserted using Golden-Gate Assembly, with no worry about the multiple illegal sites contained in it.
  
 
The firefly luciferase catalyzes a reaction that produces visible light in the 550 – 575 nm range. Numerous luc-based bioreporters have been constructed for the detection of a wide array of inorganic and organic compounds of environmental concern through the catalytic effect of the enzyme to catalysis the oxidation of firefly luciferin with the requiring of oxygen and ATP. <sup>1 </sup>
 
The firefly luciferase catalyzes a reaction that produces visible light in the 550 – 575 nm range. Numerous luc-based bioreporters have been constructed for the detection of a wide array of inorganic and organic compounds of environmental concern through the catalytic effect of the enzyme to catalysis the oxidation of firefly luciferin with the requiring of oxygen and ATP. <sup>1 </sup>
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The dual-luciferase technology means that double reporter gene is expressed simultaneously in a signal system, but independently measured by two luciferase reporter genes. One reporter gene activity is associated with specific experimental conditions for gene expression, whereas the activity of another reporter gene provides an internal control to normalize the experimental value. <sup>2 </sup>
 
The dual-luciferase technology means that double reporter gene is expressed simultaneously in a signal system, but independently measured by two luciferase reporter genes. One reporter gene activity is associated with specific experimental conditions for gene expression, whereas the activity of another reporter gene provides an internal control to normalize the experimental value. <sup>2 </sup>
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In the pmirGLO Dual-Luciferase miRNA Target Expression Vector, the luc2 is used as the primary reporter to monitor mRNA regulation and hRluc-neo acting as a control reporter for normalization and selection.
 
  
 
===Sequence and Features===
 
===Sequence and Features===

Revision as of 01:22, 2 November 2017


Fluc (with BsmBI restriction site)

This is the firefly luciferase reporter gene with a specific restriction site that could be recognized by BsmBI.

Usage and Biology

Previously, iGEM07_Ljubljana had already designed the part BBa_I712019, containing the coding sequence of Firefly Luciferase which had fulfilled function of observation in vivo.

This year,this part was improved by us to optimize its function in a dual-luciferase miRNA target expression system. Two restriction sites of BsmBI were added to the 3’ end of firefly luciferase, through which, the 3’ untranslated region of any gene can be inserted using Golden-Gate Assembly, with no worry about the multiple illegal sites contained in it.

The firefly luciferase catalyzes a reaction that produces visible light in the 550 – 575 nm range. Numerous luc-based bioreporters have been constructed for the detection of a wide array of inorganic and organic compounds of environmental concern through the catalytic effect of the enzyme to catalysis the oxidation of firefly luciferin with the requiring of oxygen and ATP. 1

The luminous intensity of luc is related to the substrate concentration, the experimental gene expression, so it can be used in the field of medical diagnostics.

The dual-luciferase technology means that double reporter gene is expressed simultaneously in a signal system, but independently measured by two luciferase reporter genes. One reporter gene activity is associated with specific experimental conditions for gene expression, whereas the activity of another reporter gene provides an internal control to normalize the experimental value. 2

Sequence and Features

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 64
    Illegal NgoMIV site found at 1408
    Illegal NgoMIV site found at 1429
    Illegal AgeI site found at 1132
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1314


Reference

[1] Fraga H, Fernandes D, Novotny J, Fontes R, Esteves da Silva JC. (Jun 2006). "Firefly luciferase produces hydrogen peroxide as a coproduct in dehydroluciferyl adenylate formation". Chembiochem.7(6):929-35. PMID: 16642538 DOI: 10.1002/cbic.200500443

[2] Heise K, Oppermann H, Meixensberger J, Gebhardt R, Gaunitz F. (May 2013)."Dual luciferase assay for secreted luciferases based on Gaussia and NanoLuc". Assay Drug Dev Technol.11(4):244-52. PMID: 23679848 DOI: 10.1089/adt.2013.509