Part:BBa_K1529302

Prhl(RL)-CmR-LasI

　We constructed this part by combining Prhl(RL) (BBa_K1529300), RBS-CmR (BBa_K395160), RBS (BBa_B0034) and LasI (BBa_C0078).
Prhl(RL) is a promoter that is activated by C4HSL.
Prhl(RL) promoter has no leak and higher expression than Prhl promoter(BBa_R0071).
On the downstream of the promoter, CmR and lasI are inserted.

　To characterize Prhl(RL)-CmR-LasI (BBa_K1529302), we introduced Prhl(RL)-CmR-LasI on pSB3K3 with Ptet-GFP-Ptet-RhlR on pSB6A1 to E. coli as “C4HSL dependent CmR and 3OC12HSL producer cell”.
In this E. coli, constitutively expressed RhlR activates the expression of CmR and LasI in the presence of C4HSL.

　To confirm C4HSL-dependent CmR production, we cultured Prhl(RL)-CmR-LasI cell in LB media with Amp, Kan and Cm.
Fig.1 shows OD590 by CmR producer cells dependent on different conditions.

Fig. 1. C4HSL-dependent CmR expressionh in 100 microg / mL chloramphenicol

　To confirm C4HSL-dependent 3OC12HSL production, we introduced Ptet-LuxR on pSB6A1 and Plux-GFP on pSB3K3 to E. coli as “Las reporter cell”.
Fig.2 shows fluorescence intensity by Rhl reporter cells dependent on different conditions.

Fig. 2. 3OC12HSL production in the presence of C4HSL

　From these experiments, we confirmed that the part Prhl(RL)-CmR-LasI (BBa_K1529302) worked accurately.
　Moreover, by co-culturing the cells containing this part with the cells containing 3OC12HSL dependent C4HSL producer part (Plux-CmR-RhlI:BBa_K1529797), we succeeded in constructing a positive feedback system. (Fig. 4.)

Fig. 3.Plasmids for the experiment of Co-culture.

Fig. 4. The growth of Campany(Prhl(RL)-CmR-LasI) and Customer(Plux-CmR-RhlI) when co-cultured for 6 hours.

Fig. 5.Increase of OD(co-culture) / OD(mono culture)

To see how the initial amount affect the growth, an additional experiment was conducted.
We conducted the experiment shown above at a smaller scale.
Increase of OD(co-culture) / OD(mono culture) was calculated by the equation shown below.
　　　OD590 of the co-cultured sample / OD590 of the mono cultured sample
　　　　*OD was measured after 4h incubation
　　　　*The ratio of customer and company in co-cultured sample is Customer : Company = 30 : 13

Sample condition is as follows.

co-cultured sample	(53, 123)	(66, 154)	(83, 192)	(104, 240)
mono cultured sample	(53, 0) + (0,123)	(66, 0) + (0,154)	(83, 0) + (0,192)	(104, 0) + (0,240)

　　　　*(Initial cell concentration of Customer[microL], Initial cells concentration of Company[microL])

When the initial amount is enough, the increase of OD(co-culture) / OD(mono culture) is high because of the mutualism between Customer and Company.
On the contrary, the ratio is low when the initial amount is small (Fig. 5).


We operated three independent co-culture assays. First, we confirmed whether mutualism was achievable. This result shows that the final OD of co-cultured cells was more than the final OD of both mono-cultured cells combined, when the initial OD of Company (Prhl(RL)-CmR-LasI:BBa_K1529302) was 0.05 (Fig. 6).

Fig. 6.First Co-culture Assay

Secondly, we specified the range of the initial OD of Customer (Plux-CmR-RhlI:BBa_K1529797) that can achieve mutualism. This result shows that the best condition for mutualism is when the initial OD of Company (Prhl(RL)-CmR-LasI:BBa_K1529302) and Customer (Plux-CmR-RhlI:BBa_K1529797) were 0.05 and 0.02 (Fig. 7).

Fig. 7.Second Co-culture Assay

Thirdly, we confirmed whether we can achieve mutualism if the initial OD became lower. This result shows that the lower the initial OD was, the less possible the mutualism could be achieved (Fig. 8).

Fig. 8.Third Co-culture Assay

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 991
  
• 1000
  COMPATIBLE WITH RFC[1000]