Difference between revisions of "Part:BBa K325210"

 
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<partinfo>BBa_K325210 parameters</partinfo>
 
<partinfo>BBa_K325210 parameters</partinfo>
 
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===Measurement by ZJU-China 2022===
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<h2>Introduction</h2>
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<p>Firefly luciferases (Luc) can catalyze the oxidation of firefly luciferin with molecular oxygen to emit light and are currently being applied as reporter genes for bioimaging and biosensors.[1] The initial reaction catalyzed by firefly luciferase (Luc) is the formation of luciferase-bound luciferyl adenylate (Luc:LH<sub>2</sub>-AMP) in the presence of Mg<sup>2+</sup> and ATP by the release of inorganic pyrophosphate (PPi). The carboxyl group of D-luciferin (LH<sub>2</sub>) is adenylated. The second step involves the oxygenation of LH<sub>2</sub>-AMP with molecular oxygen (O<sub>2</sub>) to produce the excited state of oxyluciferin (Oxyluciferin*), adenosine monophosphate (AMP) and carbon dioxide (CO<sub>2</sub>). The light emission is produced from the relaxation of excited state oxyluciferin to the corresponding ground state.</p>
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<div align="center">[[File:Ljy-measure-1.png|800px|]]</div>
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<p>Luciferin-regenerating enzyme (LRE) plays an important role in the recycling of oxyluciferin into luciferin, improving the luminescent signal of firefly luciferase, which the following two-step reaction can explain:</p>
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<p>(1) Transformation of oxyluciferin to 2-cyano-6-hydroxybenzothiazole</p>
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<p>(2) Condensation of 2-cyano-6-hydroxybenzothiazole with D-cysteine to yield luciferin. </p>
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<p>So LRE contributes to a reaction recycling oxyluciferin to D-luciferin in the presence of D-cysteine. LRE can be considered as a key enzyme to be applied in luciferase assays.[2]</p>
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<br>
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<p>Here, Luciferase and LRE are expressed, purified and characterized in <i>E.coli</i>. Finally, the kinetics and bioluminescence properties of luciferase are measured.</p>
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<h2>Materials and Methods</h2>

Revision as of 08:31, 7 October 2022


Red Firefly Luciferase and LRE
L. Cruciata
(E. coli optimised)

This part is contains the coding sequence for a mutant light-emitting enzyme (luciferase) and luciferin regenerating enzyme (LRE) from the Japanese firefly, Luciola cruciata. It will make bacteria emit light at a reddish wavelength when placed under a promoter, provided the media is supplemented with luciferin.

The luciferin regenerating enzyme will convert oxyluciferin into CHBT. CHBT is converted to more luciferin in the presence of D-cysteine.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2060
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 951
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 900
    Illegal AgeI site found at 2602
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 332
    Illegal SapI.rc site found at 2116


Measurement by ZJU-China 2022

Introduction

Firefly luciferases (Luc) can catalyze the oxidation of firefly luciferin with molecular oxygen to emit light and are currently being applied as reporter genes for bioimaging and biosensors.[1] The initial reaction catalyzed by firefly luciferase (Luc) is the formation of luciferase-bound luciferyl adenylate (Luc:LH2-AMP) in the presence of Mg2+ and ATP by the release of inorganic pyrophosphate (PPi). The carboxyl group of D-luciferin (LH2) is adenylated. The second step involves the oxygenation of LH2-AMP with molecular oxygen (O2) to produce the excited state of oxyluciferin (Oxyluciferin*), adenosine monophosphate (AMP) and carbon dioxide (CO2). The light emission is produced from the relaxation of excited state oxyluciferin to the corresponding ground state.

Ljy-measure-1.png

Luciferin-regenerating enzyme (LRE) plays an important role in the recycling of oxyluciferin into luciferin, improving the luminescent signal of firefly luciferase, which the following two-step reaction can explain:

(1) Transformation of oxyluciferin to 2-cyano-6-hydroxybenzothiazole

(2) Condensation of 2-cyano-6-hydroxybenzothiazole with D-cysteine to yield luciferin.

So LRE contributes to a reaction recycling oxyluciferin to D-luciferin in the presence of D-cysteine. LRE can be considered as a key enzyme to be applied in luciferase assays.[2]


Here, Luciferase and LRE are expressed, purified and characterized in E.coli. Finally, the kinetics and bioluminescence properties of luciferase are measured.

Materials and Methods