Difference between revisions of "Part:BBa K934022"
 
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This part produces LasI enzyme in the presence of LuxR-3OC6HSL complex.
 
This part produces LasI enzyme in the presence of LuxR-3OC6HSL complex.
  
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[[Image:Plux-LasI_result.png|thumb|center|500px|Fig. 1]]
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[[Image:Plux-LasI_result.png|thumb|center|500px|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 ([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”.  
 
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”.  
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The supernatants of the cultures of Plux-LasI cell were used as the inducer in the reporter assay.
 
The supernatants of the cultures of Plux-LasI cell were used as the inducer in the reporter assay.
  
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'''We accomplished a positive feedback system with our improved part Plux-LasI([https://parts.igem.org/Part:BBa_K934012 BBa_K934012]).'''
 
  
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[[Image:Positivefeedback.png|thumb|center|800px|This work was done by M.M, X.T and S.L.]]
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[[Image:Las_reporter_assay.png|thumb|center|500px|Fig. 2]]
  
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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.
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'''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]).'''
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[[Image:Timedependent.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 11:35, 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]