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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This system consists of a positive and a negative feedback loop. The positive feedback loop is initiated when the constitutively expressed luxR forms a complex with AHL- which is produced by the protein LuxI. The complex up-regulates the expression of luxI, GFP and AiiA, at first exponentially increasing LuxI, GFP and AiiA levels because the maturation time of LuxI is shorter than that of AiiA. Then the negative feedback loop gets the upper hand: Once maturated, the enzyme AiiA degrades AHL faster than luxI can produce it. Because GFP is under the control of the same promoter as luxI it is highly expressed until AHL is rapidly being degraded by AiiA. Once the AHL concentration is lowered enough, the expression of GFP is halted. At this point the net change in GFP will thus become negative due to the degradation. Since AiiA expression is initiated by the luxR-AHL complex, the production of AiiA also halts which causes the net change of AiiA to become negative and this eventually causes the concentration of AiiA to become low enough to allow a net production of AHL again. Since AHL is a quorum sensing molecule all cells are synchronised in their oscillatory behavior.
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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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Furthermore, this system implements two hybrid promoters: One controlling the transcriptional rate of luxI and the other one controlling the transcriptional rate of AiiA. These two promoters are respectively repressible by IPTG and aTc. Therefore, it should be possible to tune the period and the amplitude of the synchronized oscillations.
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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.
  
 
===Safety Aspects===
 
===Safety Aspects===
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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.