Difference between revisions of "Part:BBa J64010:Experience"

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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_J64010, we used PlasI(BBa_J64010)-rbs-gfp-TT on pSB3K3 as reporer part and Ptrc-rbs-lasR-TT on pBR as regulator part.  
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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_J64010, we used PlasI(BBa_J64010)-rbs-gfp-TT as reporer part and Ptrc-rbs-lasR-TT as regulator part.  
  
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Since Ptrc is a constitutive promoter, lasR is constitutively expressed and, since it is know that this lasR is a working part, the fact that the fluorescence intensity levels do not change before and after induction of BBa_K649000 by 3O-C12-HSL indicates that BBa_K649000 is not regulated by 3O-C12-HSL.
+
Since Ptrc is a constitutive promoter, lasR is constitutively expressed and, since it is know that this lasR is a working part, the fact that the fluorescence intensity levels do not change before and after induction of BBa_K649000 by 3OC12-HSL indicates that BBa_K649000 is not regulated by 3OC12-HSL.
  
  
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We improved this part. LasI promoter([https://parts.igem.org/Part:BBa_K649001:Experience BBa_K649000]) which we constructed was successfully regulated by 3O-C12-HSL.
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We improved this part. LasI promoter([https://parts.igem.org/Part:BBa_K649001:Experience BBa_K649000]) which we constructed was successfully regulated by 3OC12-HSL.
  
  
 
'''[Sample]'''
 
'''[Sample]'''
  
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pSB3K3-PlasI(BBa_J64010)-rbs-gfp-TT / pBR-Ptrc-rbs-lasR-TT
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PlasI(BBa_J64010)-rbs-gfp-TT / Ptrc-rbs-lasR-TT
  
  
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①Overnight culture of grown at 37  in LB medium containing carbenicillin and kanamycin were diluted 1:1000 in the medium, and then they were incubated at 37 °C as fresh cultures.
 
①Overnight culture of grown at 37  in LB medium containing carbenicillin and kanamycin were diluted 1:1000 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.
+
②After their OD600 reached 0.2, we added 3 µL of 500 µM 3O-C12-HSL (3OC12-HSL+) or 3µL of DMSO (3OC12-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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③After 3-hour incubation at 37 °C (OD reached approximately 1.50.), 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.
 
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.
 
|};
 
|};

Revision as of 02:28, 3 October 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_J64010

User Reviews

UNIQaa924b7fb050849f-partinfo-00000000-QINU UNIQaa924b7fb050849f-partinfo-00000001-QINU

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Tokyo-Tech iGEM 2011

Fluorescence intensity of PlasI(BBa_J64010)-gfp did not change before and after 3O-C12-HSL induction.


Effect of 3O-C12-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_J64010, we used PlasI(BBa_J64010)-rbs-gfp-TT as reporer part and Ptrc-rbs-lasR-TT as regulator part.

Since Ptrc is a constitutive promoter, lasR is constitutively expressed and, since it is know that this lasR is a working part, the fact that the fluorescence intensity levels do not change before and after induction of BBa_K649000 by 3OC12-HSL indicates that BBa_K649000 is not regulated by 3OC12-HSL.


If you want to know why the lasR is a working part, click [here] and visit our team's wiki.


We improved this part. LasI promoter(BBa_K649000) which we constructed was successfully regulated by 3OC12-HSL.


[Sample]

PlasI(BBa_J64010)-rbs-gfp-TT / Ptrc-rbs-lasR-TT


[Method]

①Overnight culture of grown at 37 in LB medium containing carbenicillin and kanamycin were diluted 1:1000 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 (3OC12-HSL+) or 3µL of DMSO (3OC12-HSL-) into the fresh cultures.

③After 3-hour incubation at 37 °C (OD reached approximately 1.50.), 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.

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