"
Team:Tokyo Tech/positivefeedback2
From 2012.igem.org
(→Materials & Methods) |
(→Protocol) |
||
| Line 30: | Line 30: | ||
JM2.300 | JM2.300 | ||
| - | + | =Protocol= | |
1. Collect liquid culture | 1. Collect liquid culture | ||
| + | |||
1.1 Prepare overnight culture of inducer cells at 37 °C for 12 hours. | 1.1 Prepare overnight culture of inducer cells at 37 °C for 12 hours. | ||
| + | |||
1.2 Take 30 μl of the overnight culture of inducer cells into LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). (→fresh culture) | 1.2 Take 30 μl of the overnight culture of inducer cells into LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). (→fresh culture) | ||
| + | |||
1.3 Incubate the flesh culture of inducer cells until the observed OD600 reaches around 0.50. Centrifuge the cells at 5000 g, 25 °C, 1 min, suspend it with 1 ml LB + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). | 1.3 Incubate the flesh culture of inducer cells until the observed OD600 reaches around 0.50. Centrifuge the cells at 5000 g, 25 °C, 1 min, suspend it with 1 ml LB + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). | ||
| + | |||
1.4 According to the Fig, take 30 μl cell suspensions into LB(3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml) + 3OC6HSL (5 nM). | 1.4 According to the Fig, take 30 μl cell suspensions into LB(3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml) + 3OC6HSL (5 nM). | ||
1.5 Incubate the inducer cells at 37 °C. | 1.5 Incubate the inducer cells at 37 °C. | ||
| + | |||
1.6 After incubating 0 ,0.5 ,1 ,2 ,4 hours, centrifuge each inducer cell at 9000 g, 4 °C, 1 min, and filter these cultured cells. | 1.6 After incubating 0 ,0.5 ,1 ,2 ,4 hours, centrifuge each inducer cell at 9000 g, 4 °C, 1 min, and filter these cultured cells. | ||
| + | |||
1.7 Samples are moved from round tube to 1.5ml tube and frozen in liquid nitrogen. | 1.7 Samples are moved from round tube to 1.5ml tube and frozen in liquid nitrogen. | ||
| + | |||
1.8 Preserve the samples at -80 °C. | 1.8 Preserve the samples at -80 °C. | ||
| + | |||
1.9 Dissolve these samples at 37 °C just before process 2.5 ,and dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30. | 1.9 Dissolve these samples at 37 °C just before process 2.5 ,and dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30. | ||
2 Reporter assay | 2 Reporter assay | ||
| + | |||
2.1 Prepare overnight culture of reporter cells at 37 °C for 12 hours. | 2.1 Prepare overnight culture of reporter cells at 37 °C for 12 hours. | ||
| + | |||
2.2 Take 30 μl of the overnight culture of reporter cells into LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). (→fresh culture) | 2.2 Take 30 μl of the overnight culture of reporter cells into LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). (→fresh culture) | ||
| + | |||
2.3 Incubate the flesh culture of reporter cells until the observed OD600 reaches around 0.50, and gather the supernatant of culture of inducer cells. | 2.3 Incubate the flesh culture of reporter cells until the observed OD600 reaches around 0.50, and gather the supernatant of culture of inducer cells. | ||
| + | |||
2.4 Centrifuge the reporter cells at 5000 g, 25 °C, 1 min, and take it into LB + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). | 2.4 Centrifuge the reporter cells at 5000 g, 25 °C, 1 min, and take it into LB + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). | ||
| + | |||
2.5 Add 30 μl samples of process 2.4 to filtrate + LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml) from process 1.9. | 2.5 Add 30 μl samples of process 2.4 to filtrate + LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml) from process 1.9. | ||
| + | |||
2.6 Incubate the reporter cells for 4 hours at 37 °C. | 2.6 Incubate the reporter cells for 4 hours at 37 °C. | ||
| + | |||
2.7 Flow cytometer measurements for GFP expression of reporter cells. | 2.7 Flow cytometer measurements for GFP expression of reporter cells. | ||
Revision as of 11:44, 23 October 2012
Contents |
Construction of the positive feedback system
Materials & Methods
Construction
A) Sender cells pSB6A1-Ptet-LuxR / pSB3K3-Plux-LasI (JM2.300)…Plux-LasI cell pSB6A1-Ptrc-LasR / pSB3K3-Plas-LuxI (JM2.300)…Plas-LuxI cell
pSB6A1-Ptet-LuxR / pSB3K3-ΔP-LasI (JM2.300)…ΔP-LasI cell
pSB6A1-Ptrc-LasR / pSB3K3-ΔP-LuxI (JM2.300)…ΔP-LuxI cell
B) Reporter cells
pSB6A1-Ptrc-LasR / pSB3K3-Plas-GFP (JM2.300)…Las reporter cell
pSB6A1-Ptet-LuxR / pSB3K3-Plux-GFP (JM2.300)…Lux reporter cell
pSB6A1-Ptrc-LasR / pSB3K3-ΔP-GFP (JM2.300)…negative control
pSB6A1-Ptrc-LasR / pSB3K3-pλ-GFP (JM2.300)…positive control
Strain
JM2.300
Protocol
1. Collect liquid culture
1.1 Prepare overnight culture of inducer cells at 37 °C for 12 hours.
1.2 Take 30 μl of the overnight culture of inducer cells into LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). (→fresh culture)
1.3 Incubate the flesh culture of inducer cells until the observed OD600 reaches around 0.50. Centrifuge the cells at 5000 g, 25 °C, 1 min, suspend it with 1 ml LB + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml).
1.4 According to the Fig, take 30 μl cell suspensions into LB(3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml) + 3OC6HSL (5 nM).
1.5 Incubate the inducer cells at 37 °C.
1.6 After incubating 0 ,0.5 ,1 ,2 ,4 hours, centrifuge each inducer cell at 9000 g, 4 °C, 1 min, and filter these cultured cells.
1.7 Samples are moved from round tube to 1.5ml tube and frozen in liquid nitrogen.
1.8 Preserve the samples at -80 °C.
1.9 Dissolve these samples at 37 °C just before process 2.5 ,and dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.
2 Reporter assay
2.1 Prepare overnight culture of reporter cells at 37 °C for 12 hours.
2.2 Take 30 μl of the overnight culture of reporter cells into LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). (→fresh culture)
2.3 Incubate the flesh culture of reporter cells until the observed OD600 reaches around 0.50, and gather the supernatant of culture of inducer cells.
2.4 Centrifuge the reporter cells at 5000 g, 25 °C, 1 min, and take it into LB + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml).
2.5 Add 30 μl samples of process 2.4 to filtrate + LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml) from process 1.9.
2.6 Incubate the reporter cells for 4 hours at 37 °C.
2.7 Flow cytometer measurements for GFP expression of reporter cells.
