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Team:Tokyo Tech/Projects/positive feedback assay/index.htm
From 2012.igem.org
Materials & Methods
Construction
A) Inducer cell pSB6A1-Ptet-LuxR / pSB3K3-Plux-LasI (JM2300)…Plux-LasI cell
pSB6A1-Ptrc-LasR / pSB3K3-Plas-LuxI (JM2300)…Plas-LuxI cell
pSB6A1-Ptet-LuxR / pSB3K3-ΔP-LasI (JM2300)…ΔP-LasI cell
pSB6A1-Ptrc-LasR / pSB3K3-ΔP-LuxI (JM2300)…ΔP-LuxI cell
B) Reporter cell
pSB6A1-Ptrc-LasR / pSB3K3-Plas-GFP (JM2300)…Las reporter cell
pSB6A1-Ptet-LuxR / pSB3K3-Plux-GFP (JM2300)…Lux reporter cell
pSB6A1-Ptrc-LasR / pSB3K3-ΔP-GFP (JM2300)…negative control
pSB6A1-Ptrc-LasR / pSB3K3-pλ-GFP (JM2300)…positive control
pSB6A1-Ptet-LuxR / pSB3K3-ΔP-GFP (JM2300)…negative control
pSB6A1-Ptet-LuxR / pSB3K3-pλ-GFP (JM2300)…positive control
2.Strain
JM2,300
3.Protocol
3OC6HSL dependent
1.collect liquid culture
1.1Prepare overnight culture of inducer cell at 37°C for 12hours.
1.2Take 30μl of the overnight culture of inducer cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml).(→fresh culture)
1.3Incubate the flesh culture of inducer cell until the observed OD600 reaches around 0.50. Centrifuge the cell at 5000g, 25°C, 1 min, suspend it with 1ml LB + antibiotics (Amp 50μg/ml).
1.4Take 30μl cell suspensions into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) + 5μM 3OC6HSL(3μl) and LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) + DMSO(3μl).
1.5Incubate the 3OC12HSL producer cells for another 4 hours at 37°C.
1.6Centrifuge the 3OC12HSL producer cells at 9000g, 4°C, 1 min, and filter the cultured cells.
1.7Dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.
2Reporter assay
2.1Prepare overnight culture of reporter cell at 37°C for 12hours.
2.2Take 30μl of the overnight culture of reporter cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
2.3Incubate the flesh culture of reporter cell until the observed OD600 reaches around 0.50, and gather the supernatant of culture of inducer cell.
2.4Centrifuge the reporter cell at 5000g, 25°C, 1 min, and take it into LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml).
2.5Add 30μl samples of process 2.4 to filtrate + LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) from process 1.7
3OC12HSL dependent
1.collect liquid culture
1.1Prepare overnight culture of inducer cell at 37°C for 12hours.
1.2Take 30μl of the overnight culture of inducer cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml).(→fresh culture)
1.3Incubate the flesh culture of inducer cell until the observed OD600 reaches around 0.50. Centrifuge the cell at 5000g, 25°C, 1 min, suspend it with 1ml LB + antibiotics (Amp 50μg/ml).
1.4Take 30μl cell suspensions into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) + 5μM 3OC6HSL(3μl) and LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) + DMSO(3μl).
1.5Incubate the 3OC12HSL producer cells for another 4 hours at 37°C.
1.6Centrifuge the 3OC12HSL producer cells at 9000g, 4°C, 1 min, and filter the cultured cells.
1.7Dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.
2Reporter assay
2.1Prepare overnight culture of reporter cell at 37°C for 12hours. 2.2Take 30μl of the overnight culture of reporter cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
2.3Incubate the flesh culture of reporter cell until the observed OD600 reaches around 0.50, and gather the supernatant of culture of inducer cell.
2.4Centrifuge the reporter cell at 5000g, 25°C, 1 min, and take it into LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml).
2.5Add 30μl samples of process 2.4 to filtrate + LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) from process 1.7
