Difference between revisions of "Part:BBa K389004"
 
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<partinfo>BBa_K389004 short</partinfo>
 
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A firefly (''Photinus pyralis'') luciferase from Promega's pGL4.10luc2 vector. Luciferase is a sensitive reporter gene.
 
 
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Structure of <em>Photinus pyralis</em> firefly luciferase. Image taken from: http://en.wikipedia.org/wiki/Luciferase.  
 
Structure of <em>Photinus pyralis</em> firefly luciferase. Image taken from: http://en.wikipedia.org/wiki/Luciferase.  
 
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A firefly (''Photinus pyralis'') luciferase from Promega's pGL4.10[luc2] vector. Luciferase is a sensitive reporter gene.
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===Usage and Biology===
 
===Usage and Biology===
 
The activity of a luciferase is measured in a luminometer - so only use this BioBrick if you have one in your lab.  
 
The activity of a luciferase is measured in a luminometer - so only use this BioBrick if you have one in your lab.  
  
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Firefly luciferase oxidizes its substrate, firefly luciferin, in presence of ATP and oxygen, emitting light (Conti ''et al.'', 1996). Because the firefly luciferin does not pass the cell membrane of ''E. coli'' under "normal" cultivation conditions the cells have to be lysated before the luciferase activity measurement.  
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Firefly luciferase oxidizes its substrate, firefly luciferin, in presence of ATP and oxygen, emitting light ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VSR-4CJB7RR-7&_user=2459438&_coverDate=03%2F31%2F1996&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=ac60aa60eaad17b2aa1c4b31f954d0a8&searchtype=a Conti ''et al.'', 1996]). Because the firefly luciferin does not pass the cell membrane of ''E. coli'' under "normal" cultivation conditions the cells have to be lysated before the luciferase activity measurement.  
  
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===Important parameters===
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{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
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|-
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!style="border-style: solid; border-width: 1px"| Experiment
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!style="border-style: solid; border-width: 1px"| Result
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|style="border-style: solid; border-width: 1px"| [[Part:BBa_K389004:Experience#Accumulation of luciferase | Behaviour during cultivation]]
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* production is growth dependent
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* degradation in stationary growth phase
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|-
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|style="border-style: solid; border-width: 1px"| [[Part:BBa_K389004:Experience#Kinetic of luciferin conversion | Kinetic of luciferin conversion]]
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|style="border-style: solid; border-width: 1px"| max. output between 20 - 40 s
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|-
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|style="border-style: solid; border-width: 1px"| [[Part:BBa_K389004:Experience#Sensitivity | Limit of detection (LOD)]]
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|style="border-style: solid; border-width: 1px"| 162 RLU ~ 0.3 % of <partinfo>J23103</partinfo> output
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|-
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|style="border-style: solid; border-width: 1px"| [[Part:BBa_K389004:Experience#Sensitivity | Limit of quantification (LOQ)]]
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|style="border-style: solid; border-width: 1px"| 306 RLU ~ 0.7 % of <partinfo>J23103</partinfo> output
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|}
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 14:57, 26 October 2010

Luciferase from pGL4.10[luc2]


Structure of Photinus pyralis firefly luciferase. Image taken from: http://en.wikipedia.org/wiki/Luciferase.
A firefly (Photinus pyralis) luciferase from Promega's pGL4.10[luc2] vector. Luciferase is a sensitive reporter gene.


Usage and Biology

The activity of a luciferase is measured in a luminometer - so only use this BioBrick if you have one in your lab.

Firefly luciferase oxidizes its substrate, firefly luciferin, in presence of ATP and oxygen, emitting light ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VSR-4CJB7RR-7&_user=2459438&_coverDate=03%2F31%2F1996&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=ac60aa60eaad17b2aa1c4b31f954d0a8&searchtype=a Conti et al., 1996]). Because the firefly luciferin does not pass the cell membrane of E. coli under "normal" cultivation conditions the cells have to be lysated before the luciferase activity measurement.


Important parameters

Experiment Result
Behaviour during cultivation
  • production is growth dependent
  • degradation in stationary growth phase
Kinetic of luciferin conversion max. output between 20 - 40 s
Limit of detection (LOD) 162 RLU ~ 0.3 % of BBa_J23103 output
Limit of quantification (LOQ) 306 RLU ~ 0.7 % of BBa_J23103 output


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