Team:Tokyo Tech/positivefeedback2

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=Construction of the positive feedback system=
=Construction of the positive feedback system=
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==Materials & Methods==
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===Construction===
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A) Sender cells
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pSB6A1-Ptet-LuxR / pSB3K3-Plux-LasI (JM2.300)…Plux-LasI cell 
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pSB6A1-Ptrc-LasR / pSB3K3-Plas-LuxI (JM2.300)…Plas-LuxI cell
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pSB6A1-Ptet-LuxR / pSB3K3-ΔP-LasI (JM2.300)…ΔP-LasI cell
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pSB6A1-Ptrc-LasR / pSB3K3-ΔP-LuxI (JM2.300)…ΔP-LuxI cell
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B) Reporter cells
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pSB6A1-Ptrc-LasR / pSB3K3-Plas-GFP (JM2.300)…Las reporter cell
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pSB6A1-Ptet-LuxR / pSB3K3-Plux-GFP (JM2.300)…Lux reporter cell
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pSB6A1-Ptrc-LasR / pSB3K3-ΔP-GFP (JM2.300)…negative control
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pSB6A1-Ptrc-LasR / pSB3K3-pλ-GFP (JM2.300)…positive control
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===Strain===
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JM2.300
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==Protocol==
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1. Collect liquid culture
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1.1 Prepare overnight culture of inducer cells at 37 °C for 12 hours.
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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).
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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).
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1.5 Incubate the inducer cells at 37 °C.
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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.
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1.7 Samples are moved from round tube to 1.5ml tube and frozen in liquid nitrogen.
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1.8 Preserve the samples at -80 °C.
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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.
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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.

Revision as of 11:42, 23 October 2012

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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.
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