Part:BBa_K1632022:Experience

Materials and Methods

1.Construction

All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.

(1) J23100_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)
(2) J23100_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)
(3) J23100_lasR_TT_promoter less_CmR (pSB6A1) + Plac_rhlI (pSB3K3)…Negative control #1
(4) J23100_lasR_TT_promoter less_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)…Negative control #2
(5) J23100_lasR_TT_Plux_CmRssrA (pSB6A1) + Plac_rhlI (pSB3K3)
(6) J23100_lasR_TT_Plux_CmRssrA (pSB6A1) + promoter less_rhlI (pSB3K3)

2.Assay protocol

1.Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2.Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5.
3.Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4.Suspend the pellet in 1mL of LB containing Amp and Kan.
5.Add 30 microL of suspension in the following medium.
a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 5 microL 3OC12HSL (3 microL) + 99.5% ethanol (3 microL)
b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + 99.5% ethanol (3 microL)
c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 5 microL 3OC12HSL (3 microL) + 100 mg/mL Chloramphenicol (3 microL)
d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + 100 mg/mL Chloramphenicol (3 microL)
6.Grow the samples of cells at 37°C for more than 8 hours.
7.Measure optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)

More information

For more information, see [http://2015.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2015 wiki].

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