Tokyo/Assay
From 2007.igem.org
Contents |
AHL assay – hybrid promoter activation by endogenous AHL
Date
2007/10/18
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
0:00 Start to prepare over night culture (1 tube for each)
12:00 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.
15:00 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
*wash
take 200 ml of culture in a Falcon tube into a eppendorf tube (1.6 or 2.0 ml tube)
centrifugation for 2 min at 9000g
discard the supernatant with a pippet
add 200 ml of PBS and dissolve the pellet
centrifugation for 2 min at 9000g
discard the supernatant with a pippet
add 200 ml of PBS and dissolve the pellet
mix well
apply 150 ml onto a well
The experiments tested yellow boxes, not gray ones.
The total volume of E. coli culture (A4Δｐ and senders) was always 400 uM.
16:00 (incubate it until the OD obs. <0.20)
17:30 stop incubation => wash* => FLA analysis（When OD reached 0.8）
