| 研究課題/領域番号 |
17K15410
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| 研究機関 | 筑波大学 |
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研究代表者 |
UTADA ANDREW 筑波大学, 生命環境系, 准教授 (90776626)
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| 研究期間 (年度) |
2017-04-01 – 2019-03-31
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| キーワード | Single-cell tracking / Applied microbiology / Microfluidics / Pseudomonas aeruginosa / mucoid |
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| 研究実績の概要 |
In H29 I began my work with the mucoid variant of Pseudomonas aeruginosa by tracking the surface motility of the mucoid variant in both flow environments and on agar plates. The first major result was to discover that the strain that I am using (mucoid variant) has a very rapid ‘reverse’ mutation where the phenotype that I am currently studying (mucoid) is lost and the strain reverts to the wild type (non-mucoid phenotype).
To aid in the analysis and to prevent data artifacts from appearing in the data analysis, I have been collaborating with colleagues to generate a reporter strain of the mucoid variant to enable easy visualization and distinction. The second major result is that we generated this mutant reporter strain, which reports whether it still maintains the mucoid phenotype or has ‘reverted’ to the wild type phenotype. It reports this change by expressing a red fluorescent protein while having the mucoid phenotype and a green fluorescent protein when it reverts to the non-mucoid phenotype.
c.The discovery of this reversion mutation and its frequency is one result and directly affects the single cell tracking of the mucoid variant. Before analyzing the single cell trajectories, I first plan to characterize the percentage of the population that expresses this reversion mutation compared to percentage that remains mucoidy. I will also measure this under different conditions to test if this phenotypic switch is controllable via the environmental conditions. Once this is complete, I plan to create a baseline based on the mixed population’s surface motility.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
This year, I established a tracking station and process work-flow by which I collect and process raw data to enable analysis of the surface motility phenomena.
The mucoid strain I am using has given a few surprises. It seems to switch phenotypes under certain conditions, which confuses the analysis because this, so called, revertant variant behaves similarly to the wild type. Therefore I have generated a fluorescent reporter strain to enable accurate distinction between the actual mucoid phenotype and not the other phenotypes. This step is crucial to achieving accurate tracking results.
I am currently optimizing the media and growth conditions to understand the when the phenotypic switch occurs.
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| 今後の研究の推進方策 |
I will complete the optimization of growth conditions to begin controlling for the phenotypic switch, using my newly generated reporter strain. Once this step is complete, I will recommence single-cell tracking to create a baseline for the mucoid variant.
From this baseline, I will begin to draw distinctions between the mucoid variant, the revertant phenotype, and wild type. The revertant phenotype is interesting because a similar reversion occurs in situ and it is important to understand if there are other differences between the revertant phenotype and wild type.
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