Difference between revisions of "Part:BBa K649001"

 
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<partinfo>BBa_K649001 short</partinfo>
 
<partinfo>BBa_K649001 short</partinfo>
  
GFP reporter regulated by 3OC12-HSL and LasR.
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Fluorescence intensity of BBa_K649001 was increased by 3OC12-HSL induction. GFP expression after induction of 3OC12-HSL is 170 times as high as before.
  
  
Fluorescence intensity of BBa_K649001 was increased by 3O-C12-HSL induction.  
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[[Image:BBa K649001 graph3.png|thumb|center|500px|Effect of 3OC12-HSL induction on fluorescence intensity<br>This work is done by Takuya Tsubaki.]]
  
  
[[Image:PlasI-GFP_experience.png|thumb|center|500px|Effect of 3O-C12-HSL induction on fluorescence intensity ]]
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Generally, in the presence of 3OC12-HSL, LasR activates lasI promoter and the transcription level of downstream gene increases, but in the absence of 3OC12-HSL, LasR can't activate lasI promoter. 2011 iGEM Tokyo-Tech characterized BBa_K649000 by means of using BBa_K649001 composed of BBa_K649000 and BBa_J54103.
  
  
Generally, in the presence of 3O-C12-HSL, lasR activates lasI promoter and the transcription level of downstream gene increases, but in the absence of 3O-C12-HSL, lasR can't activate lasI promoter. To characterize BBa_K649000, we used Ptrc-rbs-lasR-TT on pBR as regulator part. Because lasR is constitutively expressed, the difference of fluorescence intensity by 3O-C12-HSL induction indicates that BBa_K649000 is successfully regulated by 3O-C12-HSL.
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We improved previous las promoters.<br>
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<ol>
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<li>
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[https://parts.igem.org/Part:BBa_J64010:Experience our assay of BBa_J64010]
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</li>
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<li>
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[https://parts.igem.org/Part:BBa_K091117 BBa_K091117]
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</li>
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<li>
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[https://parts.igem.org/Part:BBa_R0079 BBa_R0079]
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</li>
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<li>
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[https://parts.igem.org/Part:BBa_K195616 BBa_K195616]
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</li>
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<li>
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[https://parts.igem.org/Part:BBa_J69551 BBa_J69551]
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</li>
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</ol>
  
  
'''[Sample]'''
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For more information, see [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#3. our work in Tokyo_Tech 2011 wiki].
 
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Ptrc-rbs-lasR-TT on pBR / PlasI(BBa_I649000)-rbs-gfp-TT on pSB3K3
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'''Method'''
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①Overnight cultures of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:100 in the medium, and then they were incubated at 37 °C as fresh cultures.
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②After their OD600 reached 0.2, we added 3 µL of 500 µM 3O-C12-HSL (3O-C12-HSL+) or 3µL of DMSO (3O-C12-HSL-) into the fresh cultures.
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③After 3-hour incubation at 37 °C, 0.25 mL of each culture was harvested by centrifugalization and suspended by adding 1 mL of PBS (phosphate-buffered saline).
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④We dispensed 500 µL of each suspension into a disposable tube through a cell strainer, and its fluorescence intensity was measured with a flow cytometer of Becton, Dickinson and Company.
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[https://parts.igem.org/Part:BBa_J64010:Experience our assay of BBa_J64010]
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Latest revision as of 02:47, 14 October 2011

GFP regulated by 3OC12-HSL and LasR

Fluorescence intensity of BBa_K649001 was increased by 3OC12-HSL induction. GFP expression after induction of 3OC12-HSL is 170 times as high as before.


Effect of 3OC12-HSL induction on fluorescence intensity
This work is done by Takuya Tsubaki.


Generally, in the presence of 3OC12-HSL, LasR activates lasI promoter and the transcription level of downstream gene increases, but in the absence of 3OC12-HSL, LasR can't activate lasI promoter. 2011 iGEM Tokyo-Tech characterized BBa_K649000 by means of using BBa_K649001 composed of BBa_K649000 and BBa_J54103.


We improved previous las promoters.

  1. our assay of BBa_J64010
  2. BBa_K091117
  3. BBa_R0079
  4. BBa_K195616
  5. BBa_J69551


For more information, see [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#3. our work in Tokyo_Tech 2011 wiki].


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 118
    Illegal BamHI site found at 106
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 805