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| - | {{tokyotechcss}}
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| - | <div class="whitebox">
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| - | <div id="tokyotech" style=" font:bold ;left ; font-size: 30px; color: #1E90FF; padding: 10px;">
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| - | Assays for Positive feedback system </div>
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| - | </div class="whitebox">
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| - | {{tokyotechmenubar}}
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| - | <div class="whitebox">
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| - | <div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;">
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| - | __NOTOC__
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| - | =Materials & Methods=
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| - | ==1.Construction==
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| - | [[https://2012.igem.org/Team:Tokyo_Tech/Project#Assays_for_Positive_feedback_system Back to "feedback system"]]
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| - | <div id="tokyotech" style=" font:bold ;left ; font-size: 20px; color: #1E90FF; padding: 10px;">
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| - | A) Sender cells </div>
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| - | pSB6A1-Ptet-LuxR / pSB3K3-Plux-LasI (JM2.300)…Plux-LasI cell
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| - | [[File:positivefeedbackassay13tokyotech.png|300px|center]]
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| - | pSB6A1-Ptrc-LasR / pSB3K3-Plas-LuxI (JM2.300)…Plas-LuxI cell
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| - | [[File:positivefeedbackassay14tokyotech.png|300px|center|]]
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| - | pSB6A1-Ptet-LuxR / pSB3K3-ΔP-LasI (JM2.300)…ΔP-LasI cell
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| - | [[File:positivefeedbackassay5tokyotech.png|300px|center|]]
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| - | pSB6A1-Ptrc-LasR / pSB3K3-ΔP-LuxI (JM2.300)…ΔP-LuxI cell
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| - | [[File:positivefeedbackassay6tokyotech.png|300px|center]]
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| - | <br><br><br><br><br><br>
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| - | <div id="tokyotech" style=" font:bold ;left ; font-size: 20px; color: #1E90FF; padding: 10px;">
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| - | B) Reporter cells </div>
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| - | pSB6A1-Ptrc-LasR / pSB3K3-Plas-GFP (JM2.300)…Las reporter cell
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| - | [[File:positivefeedbackassay7tokyotech.png|300px|center]]
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| - | pSB6A1-Ptet-LuxR / pSB3K3-Plux-GFP (JM2.300)…Lux reporter cell
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| - | [[File:positivefeedbackassay8tokyotech.png|300px|center]]
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| - | pSB6A1-Ptrc-LasR / pSB3K3-ΔP-GFP (JM2.300)…negative control
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| - | [[File:positivefeedbackassay9tokyotech.png|300px|center]]
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| - | pSB6A1-Ptrc-LasR / pSB3K3-pλ-GFP (JM2.300)…positive control
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| - | [[File:positivefeedbackassay10tokyotech.png|300px|center]]
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| - | pSB6A1-Ptet-LuxR / pSB3K3-ΔP-GFP (JM2.300)…negative control
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| - | [[File:positivefeedbackassay11tokyotech.png|300px|center]]
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| - | pSB6A1-Ptet-LuxR / pSB3K3-pλ-GFP (JM2.300)…positive control
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| - | [[File:positivefeedbackassay12tokyotech.png|300px|center]]
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| - | ==3.Protocol==
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| - | ===3OC6HSL-dependent 3OC12HSL production module===
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| - | [[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"]]
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| - | 1. collect liquid culture
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| - | <div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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| - | 1.1 Prepare overnight culture of inducer cell at 37°C for 12hours.
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| - | 1.2 Take 30μl of the overnight culture of inducer cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml).(→fresh culture)
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| - | 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).
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| - | 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).
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| - | 1.5 Incubate the 3OC12HSL producer cells for another 4 hours at 37°C.
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| - | 1.6 Centrifuge the 3OC12HSL producer cells at 9000g, 4°C, 1 min, and filter the cultured cells.
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| - | 1.7 Dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.
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| - | </div>
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| - | 2 Reporter assay
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| - | <div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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| - | 2.1 Prepare overnight culture of reporter cell at 37°C for 12hours.
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| - | 2.2 Take 30μl of the overnight culture of reporter cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
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| - | 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.
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| - | 2.4 Centrifuge the reporter cell at 5000g, 25°C, 1 min, and take it into LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml).
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| - | 2.5 Add 30μl samples of process 2.4 to filtrate + LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) from process 1.7
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| - | {| class="wikitable" cellpadding="2"
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| - | | align="center" style="background:#f0f0f0;"|'''3OC6HSL'''
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| - | | align="center" style="background:#f0f0f0;"|'''Plux'''
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| - | |}
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| - | 2.6 Incubate the reporter cells for 4 hours at 37°C.
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| - | 2.7 Flow cytometer measurements for GFP expression of reporter cells.
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| - | </div>
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| - | [[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"]]
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| - |
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| - | ===3OC12HSL-dependent 3OC6HSL production module===
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| - | [[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_3OC12HSL-dependent_3OC6HSL_production_module Back to "Construction of the 3OC12HSL-dependent 3OC6HSL production module"]]
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| - |
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| - | 1. collect liquid culture
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| - | <div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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| - | 1.1 Prepare overnight culture of inducer cell at 37°C for 12hours.
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| - |
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| - | 1.2 Take 30μl of the overnight culture of inducer cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml).(→fresh culture)
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| - |
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| - | 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).
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| - | 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).
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| - | 1.5 Incubate the 3OC12HSL producer cells for another 4 hours at 37°C.
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| - | 1.6 Centrifuge the 3OC12HSL producer cells at 9000g, 4°C, 1 min, and filter the cultured cells.
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| - | 1.7 Dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.
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| - | </div>
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| - | 2 Reporter assay
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| - | <div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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| - | 2.1 Prepare overnight culture of reporter cell at 37°C for 12hours.
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| - | 2.2 Take 30μl of the overnight culture of reporter cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
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| - | 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.
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| - | 2.4 Centrifuge the reporter cell at 5000g, 25°C, 1 min, and take it into LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml).
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| - | 2.5 Add 30μl samples of process 2.4 to filtrate + LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) from process 1.7
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| - |
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| - | {| class="wikitable" cellpadding="2"
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| - | | align="center" style="background:#f0f0f0;"|'''3OC12HSL'''
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| - | | align="center" style="background:#f0f0f0;"|'''Plas'''
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| - | 2.6 Induction of reporter cell for 4 hours at 37°C.
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| - | 2.7 Flow cytometer measurements for GFP expression of reporter cell.
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| - | </div>
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| - | [[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_3OC12HSL-dependent_3OC6HSL_production_module Back to "Construction of the 3OC12HSL-dependent 3OC6HSL production module"]]
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| - | ===positive feedback assay===
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| - | [[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_positive_feedback_system Back to "Construction of the positive feedback system"]]
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| - |
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| - | 1. collect liquid culture
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| - | <div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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| - | 1.1 Prepare overnight culture of inducer cells at 37°C for 12hours.
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| - |
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| - | 1.2 Take 30μl of the overnight culture of inducer cells into LB (3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
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| - |
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| - | 1.3 Incubate the flesh culture of inducer cells until the observed OD600 reaches around 0.50. Centrifuge the cells at 5000g, 25°C, 1 min, suspend it with 1ml LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml).
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| - | 1.4 According to the Fig.3-1-3-1-1, take 30μl cell suspensions into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) + 3OC6HSL (5nM) or OC6HSL(2.5nM)
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| - | [[File:positivefeedbackassay27tokyotech.png|500px|thumb|center|Fig.3-1-3-1-1]]
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| - | 1.5 Incubate the inducer cell for another 4 hours at 37°C.
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| - | 1.6 Centrifuge the inducer cell at 9000g, 4°C, 1 min, and filter the cultured cell.
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| - | 1.7 Dilute the filtrate by LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml) in 1:30.
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| - | </div>
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| - | 2 Reporter assay
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| - | <div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
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| - |
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| - | 2.1 Prepare overnight culture of reporter cells at 37°C for 12hours.
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| - |
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| - | 2.2 Take 30μl of the overnight culture of reporter cells into LB (3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
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| - |
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| - | 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.
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| - |
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| - | 2.4 Centrifuge the reporter cells at 5000g, 25°C, 1 min, and take it into LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml).
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| - | 2.5 Add 30μl samples of process 2.4 to filtrate + LB (3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml) from process 1.7
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| - | 2.6 Incubate the reporter cells for 4 hours at 37°C.
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| - | 2.7 Flow cytometer measurements for GFP expression of reporter cells.
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| - | </div>
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| - | [[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_positive_feedback_system Back to "Construction of the positive feedback system"]]
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"
