Difference between revisions of "Part:BBa K546001"
m
 
(8 intermediate revisions by 3 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K546001 short</partinfo>
 
<partinfo>BBa_K546001 short</partinfo>
  
−
The F2621 BioBrick device contains a lux pL regulated luxR repressor/activator and a lux pR promoter that is linked to an 3OC6HSL, an AHL autoinducer molecule, synthetase. The lux pL promoter yields a weak constitutive downstream expression. When LuxR and 3OC6HSL are produced, the lux pL promoter, however, will be repressed by a complex of the two. If 3OC6HSL is present in the cell, the autoinducer inactivation enzyme A (''aiiA''), with an LVA Tag, will inactivate the 3OC6HSL autoinducer molecules.
+
[[Image:Visualization_LuxR_AHL_AiiA.png|520px|thumb|A schematic representation of the BioBrick sub parts' interaction.]]
 +
This device has two protein generators:
 +
The first generator  ‘luxpL-RBS-C0062-B0015’ produces an intermediate level of the LuxR protein.  
 +
 
 +
The second generator  ‘luxpR-RBS-C0060-B0015’ has a low basal expression of the AiiA enzyme. AiiA is capable of degrading the quorum sensing molecule AHL.
 +
 
 +
It should be noted that if AHL is added to this system it forms a complex with LuxR which will increase transcription of AiiA while reducing the transcription of LuxR.
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
 +
  
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>'''Sequence and Features'''</span>
 
<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K546001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K546001 SequenceAndFeatures</partinfo>
 +
 +
 +
 +
==Protocol/Experimental data==
 +
 +
 +
Functionality of this part was tested by treating AHL mixtures with strains expressing this part. Liquid cultures were grown overnight, 1 mL aliquots of these cultures were spun down and resuspended in varying concentrations of AHL in PBS (to start the AHL degradation reaction). This was also done with a culture that contains BBa_K546002 (where aiiA is replaced with GFP).
 +
 +
After a 3h incubation, the supernatant was collected and subjected to a reporter strain: BBa_K546002. 200 µL samples of reporter strain/AHL mixtures were analyzed with a Molecular Devices Spectramax M2 spectrophotometer, detecting GFP. The result is shown below.
 +
 +
 +
[[Image: AHL_degradation_by_AiiA_reported_by_BBa_K546002correct.png]]
 +
 +
This part is functional, as AHL concentrations drop after incubation with <I>E. coli</I> transformed with this part. The GFP expression is much higher in the non treated mixtures, namely.
  
  

Latest revision as of 02:44, 22 September 2011

Lux pL controlled luxR with lux pR controlled AHL degrading enzyme AiiA(lva tag).

A schematic representation of the BioBrick sub parts' interaction.

This device has two protein generators: The first generator ‘luxpL-RBS-C0062-B0015’ produces an intermediate level of the LuxR protein.

The second generator ‘luxpR-RBS-C0060-B0015’ has a low basal expression of the AiiA enzyme. AiiA is capable of degrading the quorum sensing molecule AHL.

It should be noted that if AHL is added to this system it forms a complex with LuxR which will increase transcription of AiiA while reducing the transcription of LuxR.




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 AgeI site found at 1283
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1101


Protocol/Experimental data

Functionality of this part was tested by treating AHL mixtures with strains expressing this part. Liquid cultures were grown overnight, 1 mL aliquots of these cultures were spun down and resuspended in varying concentrations of AHL in PBS (to start the AHL degradation reaction). This was also done with a culture that contains BBa_K546002 (where aiiA is replaced with GFP).

After a 3h incubation, the supernatant was collected and subjected to a reporter strain: BBa_K546002. 200 µL samples of reporter strain/AHL mixtures were analyzed with a Molecular Devices Spectramax M2 spectrophotometer, detecting GFP. The result is shown below.


AHL degradation by AiiA reported by BBa K546002correct.png

This part is functional, as AHL concentrations drop after incubation with E. coli transformed with this part. The GFP expression is much higher in the non treated mixtures, namely.