Difference between revisions of "Part:BBa K649001"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K649001 short</partinfo>
 
<partinfo>BBa_K649001 short</partinfo>
  
 
GFP reporter regulated by 3OC12-HSL and LasR.
 
GFP reporter regulated by 3OC12-HSL and LasR.
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Fluorescence intensity of BBa_K649001 was increased by 3O-C12-HSL induction.
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[[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 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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'''[Sample]'''
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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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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 17:20, 1 October 2011

GFP regulated by 3OC12-HSL and LasR

GFP reporter regulated by 3OC12-HSL and LasR.


Fluorescence intensity of BBa_K649001 was increased by 3O-C12-HSL induction.


Effect of 3O-C12-HSL induction on fluorescence intensity


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.


[Sample]

Ptrc-rbs-lasR-TT on pBR / PlasI(BBa_I649000)-rbs-gfp-TT on pSB3K3


Method

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.

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.

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).

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.


our assay of BBa_J64010


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