From 2007.igem.org

Contents 1 AHL assay – hybrid promoter activation by endogenous AHL 1.1 Date 1.2 Purpose 1.3 Samples　 1.4 Procedure

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