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Experiment
Contents
1. Introduction |
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In the modeling (Fig. 3-1-1-1.), the better interdependence between Company and Customer requires that the expression of LasI, under the control of Prhl promoter (BBa_R0071), is the same level as the expression of RhlI, under the control of Plux promoter (BBa_R0062). Thus, we firstly tested the expression level of Prhl promoter and Plux promoter by the reporter assay (Fig. 3-1-1-2.). |
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 Fig. 3-1-1-2. Plux and Prhl Reporter Assay flow chart |
2. Summary of the experiment |
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Our purpose is to confirm actually expression levels of Prhl promoter (BBa_R0071) and Plux promoter (BBa_R0062). We prepared three plasmids sets shown in below. (Fig. 3-1-2-1.) We measured fluorescence intensity by GFP expression when we added signaling molecules. We prepared four conditions as follow.
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Fig. 3-1-2-1. Reporter plasmids
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3. Results |
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Fig. 3-1-3-1. Plux and Prhl Reporter Assay result |
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The measured activity of pre-existing Biobrick Prhl promoter (BBa_R0071) was too weak to satisfy the requirement from our modeling results. (Fig. 3-1-3-1 lane 2) In other words, the expression of RhlI under the control of Plux promoter (BBa_R0062) is higher than the expression of LasI under the control of Prhl promoter. |
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4. Materials and methods |
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4-1. Construction |
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-Strain |
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All the samples were JM2.300 strain |
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-Plasmids |
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A. Ptet-LuxR (pSB6A1), Plux-GFP (pSB3K3) |
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Fig. 3-1-4-1.
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B. Ptet-RhlR (pSB6A1), Prhl-GFP (pSB3K3) | |
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Fig. 3-1-4-2.
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4-2. Assay Protocol | |
11. Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company). | |
5. Reference |
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1. Kendall M. Gray et al. (1994) Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of Bacteriology 176(10): 3076–3080 | |
