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

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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 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>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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②After 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>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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③After 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>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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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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<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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We improved previous las promoters.<br>
 
We improved previous las promoters.<br>

Revision as of 19:58, 5 October 2011

Applications of BBa_K649001

Fluorescence intensity of BBa_K649001 was increased by 3OC12-HSL induction.


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.
      5. 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].

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