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Positive feedback assay

Materials & Methods

Construction

[Back to "feedback system"]

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

[Back to "feedback system"]

2.Strain

JM2,300

3.Protocol

3OC6HSL dependent

[Back to "Construction of the 3OC6HSL-dependent 3OC12HSL production module"]

1. collect liquid culture

1.1 Prepare overnight culture of inducer cell at 37°C for 12hours.

1.2 Take 30μl of the overnight culture of inducer cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml).(→fresh culture)

1.3 Incubate 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).

1.4 Take 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).

1.5 Incubate the 3OC12HSL producer cells for another 4 hours at 37°C.

1.6 Centrifuge the 3OC12HSL producer cells at 9000g, 4°C, 1 min, and filter the cultured cells.

1.7 Dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.

2Reporter assay

2.1 Prepare overnight culture of reporter cell at 37°C for 12hours.

2.2 Take 30μl of the overnight culture of reporter cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)

2.3 Incubate the flesh culture of reporter cell until the observed OD600 reaches around 0.50, and gather the supernatant of culture of inducer cell.

2.4 Centrifuge the reporter cell at 5000g, 25°C, 1 min, and take it into LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml).

2.5 Add 30μl samples of process 2.4 to filtrate + LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) from process 1.7

3OC6HSL	Plux
+	+
-	+
+	-
-	-

2.6 Incubate the reporter cells for 4 hours at 37℃.

2.7 Flow cytometer measurements for GFP expression of reporter cells.

[Back to "Construction of the 3OC6HSL-dependent 3OC12HSL production module"]

3OC12HSL dependent

[Back to "Construction of the 3OC12HSL-dependent 3OC6HSL production module"]

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

2.6Induction of reporter cell for 4 hours at 37°C.

2.7Flow cytometer measurements for GFP expression of reporter cell.

[Back to "Construction of the 3OC12HSL-dependent 3OC6HSL production module"]

positive feedback assay

[Back to "Construction of the positive feedback system"]

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).

1.4According to the table XXXX, take 30μl cell suspensions into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) + 3OC6HSL( nM) or OC6HSL( nM)

1.5Incubate the inducer cell for another 4 hours at 37°C.

1.6Centrifuge the inducer cell at 9000g, 4°C, 1 min, and filter the cultured cell.

1.7Dilute the filtrate by LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml) 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

2.6Induction of reporter cell for 4 hours at 37°C.

2.7Flow cytometer measurements for GFP expression of reporter cell.

[Back to "Construction of the positive feedback system"]

@@ Line 95: / Line 95: @@
 [[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_3OC6HSL-dependent_3OC12HSL_production_module Back to "Construction of the 3OC6HSL-dependent 3OC12HSL production module"]]
-.collect liquid culture
+. collect liquid culture
 <div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
-.1Prepare overnight culture of inducer cell at 37°C for 12hours.
+.1 Prepare overnight culture of inducer cell at 37°C for 12hours.
-.2Take 30μl of the overnight culture of inducer cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml).(→fresh culture)
+.2 Take 30μl of the overnight culture of inducer cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml).(→fresh culture)
-.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).
+.3 Incubate 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).
-.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).
+.4 Take 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).
-.5Incubate the 3OC12HSL producer cells for another 4 hours at 37°C.
+.5 Incubate the 3OC12HSL producer cells for another 4 hours at 37°C.
-.6Centrifuge the 3OC12HSL producer cells at 9000g, 4°C, 1 min, and filter the cultured cells.
+.6 Centrifuge the 3OC12HSL producer cells at 9000g, 4°C, 1 min, and filter the cultured cells.
-.7Dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.
+.7 Dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.
 </div>
 Reporter assay
 <div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
-.1Prepare overnight culture of reporter cell at 37°C for 12hours.
+.1 Prepare overnight culture of reporter cell at 37°C for 12hours.
-.2Take 30μl of the overnight culture of reporter cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
+.2 Take 30μl of the overnight culture of reporter cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
-.3Incubate the flesh culture of reporter cell until the observed OD600 reaches around 0.50, and gather the supernatant of culture of inducer cell.
+.3 Incubate the flesh culture of reporter cell until the observed OD600 reaches around 0.50, and gather the supernatant of culture of inducer cell.
-.4Centrifuge the reporter cell at 5000g, 25°C, 1 min, and take it into LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml).
+.4 Centrifuge the reporter cell at 5000g, 25°C, 1 min, and take it into LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml).
-.5Add 30μl samples of process 2.4 to filtrate + LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) from process 1.7
+.5 Add 30μl samples of process 2.4 to filtrate + LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) from process 1.7
+{| class="wikitable" cellpadding="2"
+| align="center" style="background:#f0f0f0;"|'''3OC6HSL'''
+| align="center" style="background:#f0f0f0;"|'''Plux'''
+|-
+| +||+
+|-
+| -||+
+|-
+| +|| -
+|-
+| -|| -
+|}
+.6	Incubate the reporter cells for 4 hours at 37℃.
+.7	Flow cytometer measurements for GFP expression of reporter cells.
 </div>

Team:Tokyo Tech/Projects/positive feedback assay/index.htm

From 2012.igem.org

Revision as of 18:56, 26 September 2012

Materials & Methods

Construction

2.Strain

3.Protocol

3OC6HSL dependent

3OC12HSL dependent

positive feedback assay