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| - | {{tokyotechcss}}
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| - | {{tokyotechtop}}
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| - | {{tokyotechmenubar}}
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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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| - | Positive feedback assay </div>
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| - | </div class="whitebox">
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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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| - |
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| - | =Materials & Methods=
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| - | ==Construction==
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| - |
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| - | [[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm Back to the mainpage]]
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| - |
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| - | A) Inducer cell
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| - |
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| - | pSB6A1-Ptet-LuxR / pSB3K3-Plux-LasI (JM2300)…Plux-LasI cell
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| - | [[File:positivefeedbackassay13tokyotech.png|200px|left]]
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| - |
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| - | <br><br><br><br><br><br>
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| - |
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| - | pSB6A1-Ptrc-LasR / pSB3K3-Plas-LuxI (JM2300)…Plas-LuxI cell
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| - | [[File:positivefeedbackassay14tokyotech.png|200px|left|]]
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| - |
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| - | <br><br><br><br><br><br>
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| - |
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| - |
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| - | pSB6A1-Ptet-LuxR / pSB3K3-ΔP-LasI (JM2300)…ΔP-LasI cell
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| - | [[File:positivefeedbackassay5tokyotech.png|200px|left|]]
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| - |
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| - | <br><br><br><br><br><br>
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| - |
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| - |
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| - |
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| - |
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| - | pSB6A1-Ptrc-LasR / pSB3K3-ΔP-LuxI (JM2300)…ΔP-LuxI cell
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| - | [[File:positivefeedbackassay6tokyotech.png|200px|left]]
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| - | <br><br><br><br><br><br>
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| - |
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| - | B) Reporter cell
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| - |
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| - | pSB6A1-Ptrc-LasR / pSB3K3-Plas-GFP (JM2300)…Las reporter cell
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| - | [[File:positivefeedbackassay7tokyotech.png|200px|left]]
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| - |
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| - | <br><br><br><br><br><br>
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| - |
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| - |
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| - | pSB6A1-Ptet-LuxR / pSB3K3-Plux-GFP (JM2300)…Lux reporter cell
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| - | [[File:positivefeedbackassay8tokyotech.png|200px|left]]
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| - | <br><br><br><br><br><br>
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| - | pSB6A1-Ptrc-LasR / pSB3K3-ΔP-GFP (JM2300)…negative control
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| - | [[File:positivefeedbackassay9tokyotech.png|200px|left]]
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| - |
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| - | <br><br><br><br><br><br>
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| - |
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| - | pSB6A1-Ptrc-LasR / pSB3K3-pλ-GFP (JM2300)…positive control
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| - | [[File:positivefeedbackassay10tokyotech.png|200px|left]]
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| - |
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| - | <br><br><br><br><br><br>
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| - |
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| - |
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| - | pSB6A1-Ptet-LuxR / pSB3K3-ΔP-GFP (JM2300)…negative control
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| - | [[File:positivefeedbackassay11tokyotech.png|200px|left]]
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| - |
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| - | <br><br><br><br><br><br>
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| - |
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| - |
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| - | pSB6A1-Ptet-LuxR / pSB3K3-pλ-GFP (JM2300)…positive control
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| - | [[File:positivefeedbackassay12tokyotech.png|200px|left]]
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| - |
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| - | <br><br><br><br><br><br>
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| - |
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| - | ==2.Strain==
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| - | JM2,300
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| - |
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| - | ==3.Protocol==
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| - | ===3OC6HSL dependent===
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| - |
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| - | 1.collect liquid culture
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| - |
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| - | 1.1Prepare overnight culture of inducer cell at 37°C for 12hours.
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| - | 1.2Take 30μl of the overnight culture of inducer cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml).(→fresh culture)
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| - | 1.3Incubate 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).
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| - |
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| - | 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).
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| - | 1.5Incubate the 3OC12HSL producer cells for another 4 hours at 37°C.
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| - | 1.6Centrifuge the 3OC12HSL producer cells at 9000g, 4°C, 1 min, and filter the cultured cells.
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| - | 1.7Dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.
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| - | 2Reporter assay
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| - | 2.1Prepare overnight culture of reporter cell at 37°C for 12hours.
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| - | 2.2Take 30μl of the overnight culture of reporter cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
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| - | 2.3Incubate 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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| - | 2.4Centrifuge 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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| - | 2.5Add 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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| - | ===3OC12HSL dependent===
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| - | 1.collect liquid culture
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| - |
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| - | 1.1Prepare overnight culture of inducer cell at 37°C for 12hours.
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| - |
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| - | 1.2Take 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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| - | 1.3Incubate 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).
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| - |
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| - | 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).
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| - |
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| - | 1.5Incubate the 3OC12HSL producer cells for another 4 hours at 37°C.
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| - | 1.6Centrifuge the 3OC12HSL producer cells at 9000g, 4°C, 1 min, and filter the cultured cells.
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| - | 1.7Dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.
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| - |
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| - | 2Reporter assay
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| - |
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| - | 2.1Prepare overnight culture of reporter cell at 37°C for 12hours.
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| - | 2.2Take 30μl of the overnight culture of reporter cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
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| - |
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| - | 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.
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| - |
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| - | 2.4Centrifuge 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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| - |
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| - | 2.5Add 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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| - | 2.6Induction of reporter cell for 4 hours at 37°C.
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| - | 2.7Flow cytometer measurements for GFP expression of reporter cell.
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| - |
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| - | ===positive feedback assay===
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| - | '''1.collect liquid culture'''
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| - |
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| - | 1.1Prepare overnight culture of inducer cell at 37°C for 12hours.
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| - |
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| - | 1.2Take 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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| - | 1.3Incubate 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).
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| - |
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| - | 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)
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| - | [[File:positivefeedbackassay16tokyotech.png|250px|right|]]
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| - | 1.5Incubate the inducer cell for another 4 hours at 37°C.
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| - |
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| - | 1.6Centrifuge the inducer cell at 9000g, 4°C, 1 min, and filter the cultured cell.
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| - |
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| - | 1.7Dilute the filtrate by LB + antibiotics (Amp 50μg/ml + Kan 30μg/ml) in 1:30.
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| - |
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| - | '''2Reporter assay'''
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| - |
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| - | 2.1Prepare overnight culture of reporter cell at 37°C for 12hours.
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| - |
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| - | 2.2Take 30μl of the overnight culture of reporter cell into LB(3ml) + antibiotics (Amp 50μg/ml + Kan 30μg/ml). (→fresh culture)
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| - |
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| - | 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.
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| - |
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| - | 2.4Centrifuge 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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| - |
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| - | 2.5Add 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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| - | 2.6Induction of reporter cell for 4 hours at 37°C.
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| - | 2.7Flow cytometer measurements for GFP expression of reporter cell.
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