Difference between revisions of "Part:BBa K934012"
 
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[[Image:Plas-LuxI_result.png|thumb|center|500px|]]
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[[Image:Plas-LuxI_result.png|thumb|center|500px|Fig. 1 This work was done by Mai Miura, Xinran Tao and Shiyue Liu.]]
  
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To characterize Plas-LuxI (BBa_K934012), we introduced Plas-LuxI (BBa_K934012) with Ptrc-LasR to E.coli as “3OC12HSL dependent 3OC6HSL producer cell”. In this E.coli, constitutively expressed LasR activates the expression of LuxI in the presence of 3OC12HSL. We then introduced Ptet-LuxR ([https://parts.igem.org/Part:BBa_S03119 BBa_S03119]) and Plux-GFP ([https://parts.igem.org/Part:BBa_K395100 BBa_K395100]) to E.coli as a “Lux reporter cell”.
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To characterize Plas-LuxI (BBa_K934012), we introduced Plas-LuxI on pSB3K3 with Ptrc-LasR on pSB6A1 to ''E.coli'' as “3OC12HSL dependent 3OC6HSL producer cell”. In this ''E.coli'', constitutively expressed LasR activates the expression of LuxI in the presence of 3OC12HSL. We then introduced Ptet-LuxR ([https://parts.igem.org/Part:BBa_S03119 BBa_S03119]) and Plux-GFP ([https://parts.igem.org/Part:BBa_K395100 BBa_K395100]) to ''E.coli'' as a “Lux reporter cell”.
  
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In the presence of 3OC6HSL produced by “3OC12HSL dependent 3OC6HSL producer cell”, GFP expression in “Lux reporter cell” was activated. Thus, the expression of GFP in “Lux reporter cell” is dually regulated by 3OC6HSL produced by “3OC12HSL dependent 3OC6HSL producer cell”, this result shows that Plas-LuxI (BBa_K934012) synthesized 3OC6HSL.
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The supernatants of the cultures of Plas-LuxI cell were used as the inducer in the reporter assay.
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[[Image:Lux_reporter_assay.png|thumb|center|500px|Fig. 2]]
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Fig. 1 shows fluorescence intensities by Lux reporter cells dependent on different conditions. Only when the supernatant of condition F was used, the fluorescence intensity of the Lux reporter cell increased while the supernatants of other three conditions did not affect. Comparing the results of the condition E and F, it can be said that with the induction of 3OC12HSL to Plas-LuxI, the fluorescence intensity of the Lux reporter cell increased by 112-folds. This result indicates that Plas-LuxI cell produced 3OC6HSL in response to 3OC12HSL induction by the function of Plas-LuxI (BBa_K934012). From this experiment, we confirmed that a improved part Plas-LuxI (BBa_K934012) worked accurately.  
  
 
We improved a previous part Plas-LuxI ([https://parts.igem.org/Part:BBa_K266000:Experience#User_Reviews BBa_K266000])  
 
We improved a previous part Plas-LuxI ([https://parts.igem.org/Part:BBa_K266000:Experience#User_Reviews BBa_K266000])  
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and accomplished a positive feedback system with our new Plux-LasI ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]).
 
  
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'''By using Plas-LuxI (BBa_K934012), we also accomplished a positive feedback system with our new part Plux-LasI ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]).'''
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[[Image:time-dependent change assay.png|thumb|center|800px||Fig. 3 This work was done by Mai Miura.]]
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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 11:36, 29 October 2012

Plas-LuxI

We constructed this part by combining BBa_K649000 and BBa_K081008. This part generates LuxI enzyme in the presence of LasR-3OC12HSL complex.


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

To characterize Plas-LuxI (BBa_K934012), we introduced Plas-LuxI on pSB3K3 with Ptrc-LasR on pSB6A1 to E.coli as “3OC12HSL dependent 3OC6HSL producer cell”. In this E.coli, constitutively expressed LasR activates the expression of LuxI in the presence of 3OC12HSL. We then introduced Ptet-LuxR (BBa_S03119) and Plux-GFP (BBa_K395100) to E.coli as a “Lux reporter cell”.

The supernatants of the cultures of Plas-LuxI cell were used as the inducer in the reporter assay.

Fig. 2

Fig. 1 shows fluorescence intensities by Lux reporter cells dependent on different conditions. Only when the supernatant of condition F was used, the fluorescence intensity of the Lux reporter cell increased while the supernatants of other three conditions did not affect. Comparing the results of the condition E and F, it can be said that with the induction of 3OC12HSL to Plas-LuxI, the fluorescence intensity of the Lux reporter cell increased by 112-folds. This result indicates that Plas-LuxI cell produced 3OC6HSL in response to 3OC12HSL induction by the function of Plas-LuxI (BBa_K934012). From this experiment, we confirmed that a improved part Plas-LuxI (BBa_K934012) worked accurately.

We improved a previous part Plas-LuxI (BBa_K266000)

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


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 749
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]