Tokyo/Activation check by cell-produced AHL

From 2007.igem.org

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

Objective:

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

Samples:

Each sample cells has two plasmids.

• Sender 1; placI LuxI on pSB1 (high copy)([http://partsregistry.org/Part:BBa_I13202 BBa_I13202]) / A4Δp ([http://partsregistry.org/Part:BBa_I751100 BBa_I751100])

• Sender 2; placI luxI on pSB4(low copy)([http://partsregistry.org/Part:BBa_I751250 BBa_I751250]) and A4Δp([http://partsregistry.org/Part:BBa_I751100 BBa_I751100])

• Sender 3; placI luxI on pBR322(low copy)([http://partsregistry.org/Part:BBa_I751350 BBa_I751350]) and A4Δp([http://partsregistry.org/Part:BBa_I751100 BBa_I751100])

• Receiver 1; (no promoter)tetR pBR322 and A4 Hybrid promoter([http://partsregistry.org/Part:BBa_I751101 BBa_I751101])

• No sender; (no promoter)luxI pBR322([http://partsregistry.org/Part:BBa_I751300 BBa_I751300]) and A4Δp([http://partsregistry.org/Part:BBa_I751100 BBa_I751100])

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）

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.