Part:BBa_K1529797

Plux-CmR-RhlI

We constructed this part by combining BBa_K395162, BBa_B0034 and BBa_C0070.

This is the relationship between auto-inducer(C4HSL, 3oxoC6HSL or 3oxoC12HSL) and regulator protein(RhlR or LuxR) as showing below.

Fig. 1 Difference of LuxR and RhlR

Both RhlR and LuxR activate the Lux promoter because DNA binding domains of RhlR and LuxR have a similar structure to each other. However, their auto-inducer binding domains are different. From this, RhlR can combine with C4HSL and 3oxoC6HSL, LuxR can combine with 3oxoC6HSL and 3oxoC12HSL.

To characterize Plux-CmR-rhlI (BBa_K1529797), we introduced Plux-CmR-rhlI on pSB3K3 with Ptet-luxR-Ptrc-RFP on pSB6A1 to E. coli as “3OC12HSL dependent CmR and C4HSL producer cell”.
In this E. coli, constitutively expressed LuxR activates the expression of CmR and RhlI in the presence of 3OC12HSL.

To confirm 3OC12HSL-dependent CmR production, we cultured Plux-CmR-rhlI cell in LB media with Amp, Kan and Cm.
Fig.3 shows OD590 by CmR producer cells dependent on different conditions. 詳しい説明

Fig. 2 タイトル.

Fig. 3 タイトル.

To confirm 3OC12HSL-dependent C4HSL production, we introduced Ptet-rhlR (BBa_K1529270) on pSB6A1 and Plux-GFP (BBa_K395100) on pSB3K3 to E. coli as “Rhl reporter cell”.
Fig.4 shows fluorescence intensity by Rhl reporter cells dependent on different conditions. 詳しい説明

Fig. 4 タイトル.

From these experiments, we confirmed that the part Plux-CmR-rhlI (BBa_K1529797) worked accurately.

Moreover, by co-culturing the cells containing this part with the cells containing C4HSL dependent 3OC12HSL producer part, Prhl(RL)-CmR-lasI(BBa_K1529302), we succeeded in constructing a positive feedback system.

Fig. 5 タイトル.

結果の説明

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