Difference between revisions of "Part:BBa K1529302"

Revision as of 11:28, 20 October 2014

Prhl(RL)-CmR-LasI

　We constructed this part by combining Prhl(RL) (BBa_K1529300), RBS-CmR (BBa_K395160), RBS (BBa_B0034) and LasI (BBa_C0078).
Prhl(RL) is a promoter that is activated by C4HSL.
Prhl(RL) promoter has no leak and higher expression than Prhl promoter(BBa_R0071).
On the downstream of the promoter, CmR and lasI are inserted.

　To characterize Prhl(RL)-CmR-LasI (BBa_K1529302), we introduced Prhl(RL)-CmR-LasI on pSB3K3 with Ptet-GFP-Ptet-RhlR on pSB6A1 to E. coli as “C4HSL dependent CmR and 3OC12HSL producer cell”.
In this E. coli, constitutively expressed RhlR activates the expression of CmR and LasI in the presence of C4HSL.

　To confirm C4HSL-dependent CmR production, we cultured Prhl(RL)-CmR-LasI cell in LB media with Amp, Kan and Cm.
Fig.1 shows OD590 by CmR producer cells dependent on different conditions.

Fig. 1. C4HSL-dependent CmR expressionh in 100 microg / mL chloramphenicol

　To confirm C4HSL-dependent 3OC12HSL production, we introduced Ptet-LuxR on pSB6A1 and Plux-GFP on pSB3K3 to E. coli as “Las reporter cell”.
Fig.2 shows fluorescence intensity by Rhl reporter cells dependent on different conditions.

Fig. 2. 3OC12HSL production in the presence of C4HSL

　From these experiments, we confirmed that the part Prhl(RL)-CmR-LasI (BBa_K1529302) worked accurately.

　Moreover, by co-culturing the cells containing this part with the cells containing 3OC12HSL dependent C4HSL producer part, Plux-CmR-RhlI(BBa_K1529797), we succeeded in constructing a positive feedback system. (Fig. 3.)

Fig. 3. The growth of Campany(Prhl(RL)-CmR-RhlI) and Customer(Plux-CmR-RhlI) when co-cultured for 6 hours.

For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2014 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 1429
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 991
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
-We constructed this part by combining Prhl(RL) (<partinfo>BBa_K1529300</partinfo>), RBS-CmR (<partinfo>BBa_K395160</partinfo>), RBS (<partinfo>BBa_B0034</partinfo>) and LasI (<partinfo>BBa_C0078</partinfo>). <br>
+　We constructed this part by combining Prhl(RL) (<partinfo>BBa_K1529300</partinfo>), RBS-CmR (<partinfo>BBa_K395160</partinfo>), RBS (<partinfo>BBa_B0034</partinfo>) and LasI (<partinfo>BBa_C0078</partinfo>). <br>
 Prhl(RL) is a promoter that is activated by C4HSL.<br>
 Prhl(RL) promoter has no leak and higher expression than Prhl promoter(<partinfo>BBa_R0071</partinfo>). <br>
 On the downstream of the promoter, CmR and lasI are inserted.<br>
 <br>
+　To characterize Prhl(RL)-CmR-LasI (<partinfo>BBa_K1529302</partinfo>), we introduced Prhl(RL)-CmR-LasI on pSB3K3 with Ptet-GFP-Ptet-RhlR on pSB6A1 to <i>E. coli</i> as “C4HSL dependent CmR and 3OC12HSL producer cell”.<br>
-To characterize Prhl(RL)-CmR-LasI (<partinfo>BBa_K1529302</partinfo>), we introduced Prhl(RL)-CmR-LasI on pSB3K3 with Ptet-GFP-Ptet-RhlR on pSB6A1 to <i>E. coli</i> as “C4HSL dependent CmR and 3OC12HSL producer cell”.<br>
 In this <i>E. coli</i>, constitutively expressed RhlR activates the expression of CmR and LasI in the presence of C4HSL. <br>
 <br>
-To confirm C4HSL-dependent CmR production, we cultured Prhl(RL)-CmR-LasI cell in LB media with Amp, Kan and Cm.<br>
+　To confirm C4HSL-dependent CmR production, we cultured Prhl(RL)-CmR-LasI cell in LB media with Amp, Kan and Cm.<br>
 Fig.1 shows OD590 by CmR producer cells dependent on different conditions.
 [[Image:C4HSL-dependent_Prhl(RL)-CmR-LasI_cell_growth.png|thumb|center|500px|<b>Fig. 1. </b>C4HSL-dependent CmR expressionh in 100 microg / mL chloramphenicol]]
 <br>
-To confirm C4HSL-dependent 3OC12HSL production, we  introduced Ptet-LuxR on pSB6A1 and Plux-GFP on pSB3K3 to <i>E. coli</i> as “Las reporter cell”.<br>
+　To confirm C4HSL-dependent 3OC12HSL production, we  introduced Ptet-LuxR on pSB6A1 and Plux-GFP on pSB3K3 to <i>E. coli</i> as “Las reporter cell”.<br>
 Fig.2 shows fluorescence intensity by Rhl reporter cells dependent on different conditions.
 [[Image:C4HSL-dependent_3OC12HSL_production.png|thumb|center|500px|<b>Fig. 2. </b>3OC12HSL production in the presence of C4HSL]]
-<br><br><br>
-From these experiments, we confirmed that the part Prhl(RL)-CmR-LasI (<partinfo>BBa_K1529302</partinfo>) worked accurately.<br>
 <br>
-Moreover, by co-culturing the cells containing this part with the cells containing 3OC12HSL dependent C4HSL producer part, Plux-CmR-RhlI(<partinfo>BBa_K1529797</partinfo>), we succeeded in constructing a positive feedback system. (Fig. 3.)
+　From these experiments, we confirmed that the part Prhl(RL)-CmR-LasI (<partinfo>BBa_K1529302</partinfo>) worked accurately.<br>
+<br><br>
+　Moreover, by co-culturing the cells containing this part with the cells containing 3OC12HSL dependent C4HSL producer part, Plux-CmR-RhlI(<partinfo>BBa_K1529797</partinfo>), we succeeded in constructing a positive feedback system. (Fig. 3.)
 [[Image:Co-culture_experiment_confirmed_Symbiosis.png|thumb|center|500px|Fig. 3. The growth of Campany(Prhl(RL)-CmR-RhlI) and Customer(Plux-CmR-RhlI) when co-cultured for 6 hours.]]
-結果の説明
 For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2014 wiki].