| 研究課題/領域番号 |
15K14608
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| 研究機関 | 首都大学東京 |
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研究代表者 |
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| 研究期間 (年度) |
2015-04-01 – 2017-03-31
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| キーワード | Electron Transfer / Heme Proteins / Syntrophy / Methane oxidation / Electron Microscopy / Antibodies / surface proteins |
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| 研究実績の概要 |
We previously demonstrated that electronic conductivity can be seen by conducting activity analyses of individual cells within consortia (McGlynn et alia 2015, Nature). We also demonstrated that the interactions occurring between species in these consortia could be decoupled by adding soluble electron acceptors (Scheller et alia 2016 Science). Based off of these observations, we identified the enzymes needed to perform anaerobic methane oxidation in archaea in general (McGlynn in Prep).
It is likely position of multi-heme cytochromes which are positioned on the outside of the archaeal cells. It is known that these archaea genetically encode a number of potential proteins. Prior to performing the antibody studies described in our proposal, we therefor tested to see which of these are actually expressed. We found that all the relevant protein coding genes are expressed, and we also found that addition of AQDS (a soluble electron acceptor that decouples the electronic interaction between cells) resulted in a massive down regulation of these transcripts. These results in total demonstrate that these cytochrome proteins have a specific functional role in the process of anaerobic methane oxidation.
In addition to this work, we investigated the 3 dimensional structure of the protein layer which occurs on the outside of ANME archaea. Here, we used transmission electron microscopy with tilt tomography were able to demonstrate that the potential protein layer appears to be of near uniform thickness on the outside of the cell.
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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
The main goal of this research is to determine precisely which proteins are involved in direct interspecies electron transfer occurring in syntrophic methane oxidizing communities. Currently, we have demonstrated i) that the protein coating surrounding cells appears uniform across the cell surface and ii) the proteins we identified in ANME-archaea genomes are expressed (by using transcriptomics). The next challenge is to either express these proteins and then generate antibodies, or alternatively generate antibodies against peptides. We are currently in the process of synthesizing antibodies which will target these proteins specifically, so we can determine where exactly these proteins are located.
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| 今後の研究の推進方策 |
We will clone and express the genes that we now know are expressed and attempt purification followed by the generation of polyclonal antibodies and TEM localization of the peptides. At the same time, we will pursue the generation of antibodies that are generated against synthetic peptides of the expressed proteins.
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| 次年度使用額が生じた理由 |
This year we did not spend the entire proposed amount, because I was waiting until the transcriptomics work was complete. Now that we know exactly which proteins are being expressed, we will use the amount of the grant to generate antibodies as initially proposed in this research. Waiting until now gives us the confidence to proceed with the planned work.
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| 次年度使用額の使用計画 |
We will use the budget as initially described, generating antibodies against purified proteins. We will also use a portion of the budget to generate antibodies against synthetic peptide fragments of the target proteins. Using these two approaches, we will maximize our chances of obtaining results observable by transmission electron microscopy.
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