Difference between revisions of "Part:BBa K649001"

Latest revision as of 02:47, 14 October 2011

GFP regulated by 3OC12-HSL and LasR

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. 2011 iGEM Tokyo-Tech characterized BBa_K649000 by means of using BBa_K649001 composed of BBa_K649000 and BBa_J54103.

We improved previous las promoters.

For more information, see [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#3. our work in Tokyo_Tech 2011 wiki].

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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K649001 short</partinfo>
-GFP reporter regulated by 3OC12-HSL and LasR.
+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.
-Fluorescence intensity of BBa_K649001 was increased by 3O-C12-HSL induction.
+[[Image:BBa K649001 graph3.png|thumb|center|500px|Effect of 3OC12-HSL induction on fluorescence intensity<br>This work is done by Takuya Tsubaki.]]
-[[Image:PlasI-GFP_experience.png|thumb|center|500px|Effect of 3O-C12-HSL induction on fluorescence intensity ]]
+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. 2011 iGEM Tokyo-Tech characterized BBa_K649000 by means of using BBa_K649001 composed of BBa_K649000 and BBa_J54103.
-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.
+We improved previous las promoters.<br>
+<ol>
+<li>
+[https://parts.igem.org/Part:BBa_J64010:Experience our assay of BBa_J64010]
+</li>
+<li>
+[https://parts.igem.org/Part:BBa_K091117 BBa_K091117]
+</li>
+<li>
+[https://parts.igem.org/Part:BBa_R0079 BBa_R0079]
+</li>
+<li>
+[https://parts.igem.org/Part:BBa_K195616 BBa_K195616]
+</li>
+<li>
+[https://parts.igem.org/Part:BBa_J69551 BBa_J69551]
+</li>
+</ol>
-'''[Sample]'''
+For more information, see [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#3. our work in Tokyo_Tech 2011 wiki].
-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.
-[https://parts.igem.org/Part:BBa_J64010:Experience our assay of BBa_J64010]