Difference between revisions of "Part:BBa K546005"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K546005 short</partinfo>
 
<partinfo>BBa_K546005 short</partinfo>
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This BioBrick composite part has an, colony wide, initially increasing and finally constant Green Fluorescent Protein (GFP) with a LVA Tag output.
 
  
  
 
===Usage and Biology===
 
===Usage and Biology===
  
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LuxR is slightly produced by the sub-[https://parts.igem.org/Part:BBa_K546003 BioBrick part] K546003 at the beginning. Meanwhile also the mildly transcribed BioBrick part C0061 creates an increase in the [http://en.wikipedia.org/wiki/Autoinducer autoinducer] 3OC6HSL. As the LuxR and the autoinducer are formed simultaneously, a complex of these two forms -in all the cells of a colony. This complex initiates a positive and two times a negetive feedback loop. The gene luxI is part of the positive feedback loop and will be transcribed more often (resulting in a greater autoinducer production), but the promoter in front of the luxr gene will down regulate the formation of LuxR. The increase in and finally more or less equilibrium state of the LuxR-autoinducer complex is monitored by the Green Fluorescent Protein with a LVA Tag.
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[[Image:Visualization_LuxR_LuxI_GFP.png|520px|thumb|A schematic representation of the BioBrick sub parts' interaction.]]
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This device has three protein generators:
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lux pl-RBS-C0062-B0015 produces LuxR. LuxR forms a complex with the quorum sensing molecule AHL and this complex subsequently increases the transcriptional rate of the lux pR promoter while also decreasing the transcriptional rate of the lux pL promoter. Both of the other generators are under control of this lux pR promoter.
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The second generator  ‘luxpR-RBS-J04031-B0015’has a low basal expression of the GFP, which can - of course - be monitored.
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The third generator produces luxI, which produces the quorum sensing molecule AHL. In combination with LuxR, AHL thus increases the production of AHL and GFP.
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===Safety Aspects===
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This BioBrick part produces an autoinducer that might interact with  [http://2011.igem.org/Team:Wageningen_UR/Safety/One#Risk_Identification_of_BioBrick_System_Inside_the_Cell_Chassis some pathogens].
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<span class='h3bb'>'''Sequence and Features'''</span>
 
<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K546005 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K546005 SequenceAndFeatures</partinfo>
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==Experimental data==
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This part was tested by measuring overnight <I>E. coli</I> cultures transformed with high copy plasmid pSB1A2 containing this insert. Fluorescence was measured in a Molecular Devices Spectramax M2 spectrophotometer, exciting the samples at 485 nm and detecting at 510 nm. 200 µL samples were analyzed in an opaque 96-wells plate. The GFP expression was monitored for 2 hours, with BBa_K546002 transformants (high copy, pSB1A2) as negative control.
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The outcome is shown below. R0063-F2621 refers to the repaired version of F2621, submitted as [https://parts.igem.org/Part:BBa_K546003 K546003].
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[[Image:Fluorescence_measurement_BBa_K546005-BBa_K546002correct.png]]
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GFP expression rapidly increases over time, indicating a functional positive feedback loop.
  
  

Latest revision as of 02:43, 22 September 2011

Lux pL controlled LuxR + lux pR autoinducing LuxI (lva tag) + lux pR controlled GFP(lva tag).


Usage and Biology

A schematic representation of the BioBrick sub parts' interaction.

This device has three protein generators: lux pl-RBS-C0062-B0015 produces LuxR. LuxR forms a complex with the quorum sensing molecule AHL and this complex subsequently increases the transcriptional rate of the lux pR promoter while also decreasing the transcriptional rate of the lux pL promoter. Both of the other generators are under control of this lux pR promoter.

The second generator ‘luxpR-RBS-J04031-B0015’has a low basal expression of the GFP, which can - of course - be monitored.

The third generator produces luxI, which produces the quorum sensing molecule AHL. In combination with LuxR, AHL thus increases the production of AHL and GFP.

Safety Aspects

This BioBrick part produces an autoinducer that might interact with [http://2011.igem.org/Team:Wageningen_UR/Safety/One#Risk_Identification_of_BioBrick_System_Inside_the_Cell_Chassis some pathogens].


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 3909
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1101
    Illegal BsaI.rc site found at 1828
    Illegal BsaI.rc site found at 2086
    Illegal BsaI.rc site found at 3189


Experimental data

This part was tested by measuring overnight E. coli cultures transformed with high copy plasmid pSB1A2 containing this insert. Fluorescence was measured in a Molecular Devices Spectramax M2 spectrophotometer, exciting the samples at 485 nm and detecting at 510 nm. 200 µL samples were analyzed in an opaque 96-wells plate. The GFP expression was monitored for 2 hours, with BBa_K546002 transformants (high copy, pSB1A2) as negative control.

The outcome is shown below. R0063-F2621 refers to the repaired version of F2621, submitted as K546003.

Fluorescence measurement BBa K546005-BBa K546002correct.png

GFP expression rapidly increases over time, indicating a functional positive feedback loop.