Difference between revisions of "Part:BBa K649001:Experience"

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__NOTOC__
 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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how you used this part and how it worked out.
 
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===Applications of BBa_K649001===
 
===Applications of BBa_K649001===
  
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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.
  
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[[Image:K649001_experience.png|thumb|center|400px|Effect of 3O-C12-HSL induction on fluorescence intensity ]]
 
  
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[[Image:BBa K649001 graph3.png|thumb|right|500px|Effect of 3OC12-HSL induction on fluorescence intensity<br>This work is done by Takuya Tsubaki.]]
  
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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. To characterize BBa_K649000, we used Ptrc-rbs-lasR-TT as regulator part. Because LasR is constitutively expressed, the difference of fluorescence intensity by 3OC12-HSL induction indicates that BBa_K649000 is successfully regulated by 3OC12-HSL.
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'''[Sample]'''
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Ptrc-rbs-lasR-TT / PlasI(BBa_I649000)-rbs-gfp-TT
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Ptrc-rbs-lasR-TT / Promoterless-rbs-gfp-TT (negative control)
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'''[Method]'''
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<ol>
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<li>Overnight cultures of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:100 in the medium, and overnight cultures of promoterless negative control strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:200 in the medium , and then they were incubated at 37 °C as fresh cultures.
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</li>
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<li>After their OD600 reached 0.2, we added 3 µL of 500 µM 3OC12-HSL (3OC12-HSL+) or 3 µL of DMSO (3OC12-HSL-) into the fresh cultures.
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</li>
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<li>After 3-hour incubation at 37 °C (OD reached approximately 1.80.), 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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</li>
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<li>We 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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</li>
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</ol>
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We improved previous las promoters.<br>
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<ol>
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<li>
 
[https://parts.igem.org/Part:BBa_J64010:Experience our assay of BBa_J64010]
 
[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>
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To prove that the LasR regulator used in our lasI prmoter assay works, we did another assay. Details about this assay can be found [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#1. here].
  
 
===User Reviews===
 
===User Reviews===
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<!-- DON'T DELETE --><partinfo>BBa_K649001 EndReviews</partinfo>
 
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Latest revision as of 15:52, 27 September 2013

Applications of BBa_K649001

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. To characterize BBa_K649000, we used Ptrc-rbs-lasR-TT as regulator part. Because LasR is constitutively expressed, the difference of fluorescence intensity by 3OC12-HSL induction indicates that BBa_K649000 is successfully regulated by 3OC12-HSL.


[Sample]

Ptrc-rbs-lasR-TT / PlasI(BBa_I649000)-rbs-gfp-TT

Ptrc-rbs-lasR-TT / Promoterless-rbs-gfp-TT (negative control)


[Method]

  1. Overnight cultures of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:100 in the medium, and overnight cultures of promoterless negative control strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:200 in the medium , and then they were incubated at 37 °C as fresh cultures.
  2. After their OD600 reached 0.2, we added 3 µL of 500 µM 3OC12-HSL (3OC12-HSL+) or 3 µL of DMSO (3OC12-HSL-) into the fresh cultures.
  3. After 3-hour incubation at 37 °C (OD reached approximately 1.80.), 0.25 mL of each culture was harvested by centrifugalization and suspended by adding 1 mL of PBS (phosphate-buffered saline).
  4. We 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.

We improved previous las promoters.

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


To prove that the LasR regulator used in our lasI prmoter assay works, we did another assay. Details about this assay can be found [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#1. here].

User Reviews

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