Tokyo/Activation check by cell-produced AHL


Works top  0.Hybrid promoter  1.Formulation  2.Assay1  3.Simulation  4.Assay2  5.Future works

Activation check by cell-produced AHL   Expression level check on promoters + plasmid sets of A and B sides

Activation check by cell-produced AHL


To check if worker E. coli (Sender) can produce enough AHL for our model to work by using different copy numbers of plasmids.


Each sample cells has two plasmids.

  • Receiver 1; (no promoter)tetR pBR322 and A4 Hybrid promoter(BBa_I751101)


Start to prepare over night culture (1 tube for each)
Make fresh culture with 3 ul of the overnight culture in 3ml of LB + Amp + Kan (LBAK) (3 tubes for each)
Incubation in a shaker until the observed OD (ODobs) reaches up to 0.2
Equalize ODobs of all the samples by adding LB media.
Centrifugation at 5000 rpm and wash out the LB media.

Add cells with A4Δp and each of the following to 2.4 ml of LBAK

Table 1. Volume of AHL senders and receivers
A yellow box represents the sets of a sender (or AHL solution as a control) and a receiver at an indicated volume used in this experiment. A black box represents that not tested here.

The total volume of E. coli culture (A4Δp and senders) was always 400 uM.
(incubate it until the OD obs. <0.20)

Stop incubation => Wash => FLA analysis(When OD reached 0.8)

Result & Conclusion:

Fig.1 Result By culturing with AHL producing cells, the lux lac hybrid promoter in receiver cell was activated without externally adding AHL. Regardless of the copy numbers of the luxI encoding plasmids, the hybrid promoter was activated.

Not only a high-copy-number-plasmid pSB1 derivative, but also low copy number plasmid pSB4- and pBR derivatives produced enough AHL to activate the LacI hybrid promoter in other cells. Especially, the pBR derivative planned to use in the current construction, remarkably produced AHL in the present experiments.