Difference between revisions of "Part:BBa K415000"
 
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<partinfo>BBa_K415000 short</partinfo>
 
<partinfo>BBa_K415000 short</partinfo>
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BBa_K415000 represents an AND-Gate promoter controlling the expression of expressing mCherry. The promoter requires two inputs: an environment free of cI and secondly, quorum sensing, in the form of LuxR-homoserine-lactone for triggering the pLux promoter.
  
 
<partinfo>BBa_K415000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K415000 SequenceAndFeatures</partinfo>
  
  
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BBa_K415000 represents an AND-Gate promoter controlling the expression of expressing mCherry. The promoter requires two inputs: an environment free of cI and secondly, quorum sensing, in the form of LuxR-homoserine-lactone for triggering the pLux promoter. The part was constructed via the insertion of BBa_J06702 into a BBa_R0065 backbone(pSB1A2). The following 1% agarose gel, prepared from powder and 1% TAE and stained with SYBR® Safe DNA gel stain, presents confirmation of said construction. The part was run along the standard of Hyperladder 1.  
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Confirmation: The part was constructed via the insertion of BBa_J06702 into BBa_R0065 which has the backbone pSB1A2. The following 1% agarose gel, prepared from agarose mix and 1% TAE and stained with SYBR® Safe DNA gel stain, presents confirmation of said construction. The part was run along the standard of Hyperladder 1.  
  
 
Figure 1
 
Figure 1
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The fluorescence of mCherry was then quantified using FACs(Fluorescence-activated cell sorting) running two samples: -AHL and +AHL. Negative control of plasmid pRCV3(Plux-GFP) was used to ensure adequate concentrations of AHL induction. The addition of AHL tests whether or not the circuit will respond without LuxR. The fluorescence of mCherry was measured in arbitrary units.  
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The fluorescence of mCherry was then quantified using FACs(Fluorescence-activated cell sorting) running two samples: -AHL and +AHL. The samples were grown overnight in 4ml of LB with +/- 130uM AHL and Amp antibiotic in a 37C incubator with dh5-alpha cells.
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Plasmid pRCV3(Plux-GFP) was used as control to ensure adequate concentrations of AHL induction. The addition of AHL tests whether or not the circuit will respond without LuxR. The fluorescence of mCherry was measured in arbitrary units.  
  
 
Figure 3
 
Figure 3
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It is clear that the circuit does not respond to the addition of AHL. The following are FACs sheets:
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It is clear from the above figure that the average mCherry fluorescence and thus this circuit does not respond to the addition of AHL. The following are FACs sheets:
  
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Figure 4: -AHL
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Figure 4: K415000 -AHL
 
[[Image:K415000Basal.jpg|center|500px]]
 
[[Image:K415000Basal.jpg|center|500px]]
  
  
  
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Figure 5: +AHL
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Figure 5: K415000 +AHL
 
[[Image:K415000+AHL facs.jpg|center|500px]]
 
[[Image:K415000+AHL facs.jpg|center|500px]]
  

Latest revision as of 01:46, 27 October 2010

pLux/cI-OR : RBS-mCherry : Term

BBa_K415000 represents an AND-Gate promoter controlling the expression of expressing mCherry. The promoter requires two inputs: an environment free of cI and secondly, quorum sensing, in the form of LuxR-homoserine-lactone for triggering the pLux promoter.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Confirmation: The part was constructed via the insertion of BBa_J06702 into BBa_R0065 which has the backbone pSB1A2. The following 1% agarose gel, prepared from agarose mix and 1% TAE and stained with SYBR® Safe DNA gel stain, presents confirmation of said construction. The part was run along the standard of Hyperladder 1.

Figure 1

K415000 gel.jpg


The following is a plasmid map of the part in the pSB1A2 backbone:

Figure 2

K415000 geneious.jpg


The fluorescence of mCherry was then quantified using FACs(Fluorescence-activated cell sorting) running two samples: -AHL and +AHL. The samples were grown overnight in 4ml of LB with +/- 130uM AHL and Amp antibiotic in a 37C incubator with dh5-alpha cells. Plasmid pRCV3(Plux-GFP) was used as control to ensure adequate concentrations of AHL induction. The addition of AHL tests whether or not the circuit will respond without LuxR. The fluorescence of mCherry was measured in arbitrary units.

Figure 3

K415000.jpg


It is clear from the above figure that the average mCherry fluorescence and thus this circuit does not respond to the addition of AHL. The following are FACs sheets:

Figure 4: K415000 -AHL

K415000Basal.jpg


Figure 5: K415000 +AHL

K415000+AHL facs.jpg



Source

https://parts.igem.org/wiki/index.php?title=Part:BBa_R0065

https://parts.igem.org/wiki/index.php?title=Part:BBa_J06702

References