Difference between revisions of "Part:BBa J64010:Experience"
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<I>Tokyo-Tech iGEM 2011</I> | <I>Tokyo-Tech iGEM 2011</I> | ||
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| − | Fluorescence intensity of PlasI(BBa_J64010)-gfp did not change before and after | + | Fluorescence intensity of PlasI(BBa_J64010)-gfp did not change before and after 3OC12-HSL induction. |
| − | [[ | + | [[Image:BBa J64010 graph3.png|thumb|center|400px|Effect of 3OC12-HSL induction on fluorescence intensity<br>This work is done by Takuya Tsubaki.]] |
| − | This work is done by Takuya Tsubaki.]] | + | |
| − | Generally, in the presence of | + | 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 | + | 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. |
| − | |||
| + | 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. | ||
| + | |||
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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]. | ||
| − | |||
'''[Sample]''' | '''[Sample]''' | ||
| − | + | PlasI(BBa_J64010)-rbs-gfp-TT / Ptrc-rbs-lasR-TT | |
| + | |||
| + | promoterless-gfp / Ptrc-rbs-lasR-TT-PlasI-rbs-cI-TT (negative control) | ||
'''[Method]''' | '''[Method]''' | ||
| − | ①Overnight culture of grown at 37 | + | ①Overnight culture of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin, and overnight culture of promoterless negative control grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:300 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 | + | ②After their OD600 reached 0.2, we added 3 µL of 500 µM 3OC12-HSL (3OC12-HSL+) or 3µL of DMSO (3OC12-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). | + | ③After 3-hour incubation at 37 °C (OD approximately reached 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. | ||
|}; | |}; | ||
Latest revision as of 03:55, 5 October 2011
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Tokyo-Tech iGEM 2011 |
Fluorescence intensity of PlasI(BBa_J64010)-gfp did not change before and after 3OC12-HSL induction.
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.
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 promoterless-gfp / Ptrc-rbs-lasR-TT-PlasI-rbs-cI-TT (negative control)
①Overnight culture of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin, and overnight culture of promoterless negative control grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:300 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 3OC12-HSL (3OC12-HSL+) or 3µL of DMSO (3OC12-HSL-) into the fresh cultures. ③After 3-hour incubation at 37 °C (OD approximately reached 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. |
