Tokyo/Activation check by cell-produced AHL

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Revision as of 14:08, 25 October 2007

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

Purpose

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

Samples　

placI luxI on PSB1(high copy) and A4Δp(Sender 1)
placI luxI on PSB4(low copy) and A4Δp(Sender 2)
placI luxI on PBR322(low copy) and A4Δp(Sender 3)
(no promoter)tetR pBR322 and A4 Hybrid promoter(Receiver 1)
(no promoter)luxI pBR322 and A4Δp

Procedure

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 recievers
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.
Wash
(incubate it until the OD obs. <0.20)

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

Results Conclusion

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

Not only high copy number plasmid pSB1, also low copy number plasmid pSB4 and pBR produced enough AHL to activate the LacI hybrid promoter in other cells. Especially, pSB４ remarkably produced AHL in the present experiment.