Part:BBa_K934022

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

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

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]