Difference between revisions of "Part:BBa K934022"
 
(14 intermediate revisions by 2 users not shown)
Line 5: Line 5:
 
This part produces LasI enzyme in the presence of LuxR-3OC6HSL complex.
 
This part produces LasI enzyme in the presence of LuxR-3OC6HSL complex.
  
−
[[Image:Plux-LasI_result.png|thumb|center|500px|]]
+
[[Image:Plux-LasI_result.png|thumb|center|500px|Fig. 1 This work was done by Mai Miura, Xinran Tao and Shiyue Liu.]]
  
−
In the presence of 3OC12HSL produced by “3OC6HSL dependent 3OC12HSL producer cell”, “Las reporter cell” was activated and GFP was expressed. Thus, the expression of GFP in “Las reporter cell” is dually regulated by 3OC12HSL produced by “3OC6HSL dependent 3OC12HSL producer cell”, this result shows that Plux-lasI(K934022) synthesized 3OC12HSL.
+
To characterize Plux-LasI (BBa_K934022), we introduced Plux-LasI pSB3K3 with Ptet-LuxR ([https://parts.igem.org/Part:BBa_S03119 BBa_S03119]) on pSB6A1 to ''E.coli'' as “3OC6HSL dependent 3OC12HSL producer cell”. In this ''E.coli'', constitutively expressed LuxR activates the expression of LasI in the presence of 3OC6HSL. We then introduced Ptrc-LasR  and Plas-GFP ([https://parts.igem.org/Part:BBa_K649001 BBa_K649001]) to ''E.coli'' as a “Las reporter cell”.  
  
−
We accomplished a positive feedback system with our improved part Plux-LasI([https://parts.igem.org/Part:BBa_K934012 BBa_K934012]).
+
The supernatants of the cultures of Plux-LasI cell were used as the inducer in the reporter assay.
 +
 
 +
 
 +
[[Image:Las_reporter_assay.png|thumb|center|500px|Fig. 2]]
 +
 
 +
Fig. 1 shows fluorescence intensities by Las reporter cells dependent on different conditions. Only when the supernatant of condition B was used, the fluorescence intensity of the Las reporter cell increased, while the supernatants of other three conditions did not affect. Comparing the results of the condition A and B, it can be said that with the induction of 3OC6HSL to Plux-LasI cell, the fluorescence intensity of the Las reporter cell increased by 20-folds. This result indicates that Plux-LasI cell produced 3OC12HSL in response to 3OC6HSL induction by the function of Plux-LasI (BBa_934022).From this experiment, we confirmed that a new part Plux-LasI (BBa_K934022) worked accurately.
 +
 
 +
'''By using Plux-LasI (BBa_K934022), we also accomplished a positive feedback system with our improved part Plux-LasI([https://parts.igem.org/Part:BBa_K934012 BBa_K934012]).'''
 +
 
 +
[[Image:Timedependent.png|thumb|center|800px|Fig. 3 This work was done by Mai Miura.]]
 +
 
 +
As compared red solid line with blue dotted line in the condition i (both Plux-LasI cell and Plas-LuxI cell coexist), the Fig. 3 shows that the fluorescence intensity of Las reporter increases at first (0-1h), and then that of Lux reporter starts to increase (1-2h). This result indicates that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL, whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level. This behavior strongly suggests the appearance of the positive feedback.
  
 
For more information, see [http://2012.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2012 wiki].
 
For more information, see [http://2012.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2012 wiki].

Latest revision as of 11:35, 29 October 2012

Plux-LasI

We constructed this part by combining BBa_R0062 and BBa_K081016. This part produces LasI enzyme in the presence of LuxR-3OC6HSL complex.

Fig. 1 This work was done by Mai Miura, Xinran Tao and Shiyue Liu.

To characterize Plux-LasI (BBa_K934022), we introduced Plux-LasI pSB3K3 with Ptet-LuxR (BBa_S03119) on pSB6A1 to E.coli as “3OC6HSL dependent 3OC12HSL producer cell”. In this E.coli, constitutively expressed LuxR activates the expression of LasI in the presence of 3OC6HSL. We then introduced Ptrc-LasR and Plas-GFP (BBa_K649001) to E.coli as a “Las reporter cell”.

The supernatants of the cultures of Plux-LasI cell were used as the inducer in the reporter assay.


Fig. 2

Fig. 1 shows fluorescence intensities by Las reporter cells dependent on different conditions. Only when the supernatant of condition B was used, the fluorescence intensity of the Las reporter cell increased, while the supernatants of other three conditions did not affect. Comparing the results of the condition A and B, it can be said that with the induction of 3OC6HSL to Plux-LasI cell, the fluorescence intensity of the Las reporter cell increased by 20-folds. This result indicates that Plux-LasI cell produced 3OC12HSL in response to 3OC6HSL induction by the function of Plux-LasI (BBa_934022).From this experiment, we confirmed that a new part Plux-LasI (BBa_K934022) worked accurately.

By using Plux-LasI (BBa_K934022), we also accomplished a positive feedback system with our improved part Plux-LasI(BBa_K934012).

Fig. 3 This work was done by Mai Miura.

As compared red solid line with blue dotted line in the condition i (both Plux-LasI cell and Plas-LuxI cell coexist), the Fig. 3 shows that the fluorescence intensity of Las reporter increases at first (0-1h), and then that of Lux reporter starts to increase (1-2h). This result indicates that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL, whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level. This behavior strongly suggests the appearance of the positive feedback.

For more information, see [http://2012.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2012 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 745
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 307
  • 1000
    COMPATIBLE WITH RFC[1000]