| 研究課題/領域番号 |
15K16558
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| 研究機関 | 東京大学 |
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研究代表者 |
パシオラ トビー 東京大学, 理学(系)研究科(研究院), 助教 (60750239)
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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 | peptide / antibiotic |
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| 研究実績の概要 |
This project involves the development of a gel display screening system for the discovery of anti-microbial cyclic peptides from genetically reprogrammed peptide libraries. Areas originally proposed for completion in the first year were: 1) optimisation of gel casting conditions, 2) optimisation of in-gel enzymatic steps, 3) assessment of required local DNA concentration and 4) determination of appropriate cell growth conditions.
Goals 1) and 2) have been completed as planned, with synthesis of a fluorescent protein by transcription and translation from a DNA template confirmed in-gel under optimised conditions. Goal 3) has also been completed although, as anticipated, local DNA concentration was found to be insufficient for screening purposes and the development of a technique for the generation of micro-beads coated with clonal copies of a single DNA molecule was required. This has been completed successfully and transcription and translation from such beads in-gel has been confirmed. Goal 4) has also been achieved using a soft-agar overlay technique to produce a bacterial monolayer.
However, whilst each component of the screening system now appears to be in place, final validation of the screening system using a positive control antimicrobial peptide has not been achieved. This is due to the fact that known anti-microbial peptides are not sufficiently potent for this purpose, as discussed in the following sections of this report.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
As described above, known anti-microbial peptides proved to lack sufficient potency for final validation of the screening system (of the peptides tested, none were active below 25 micromolar with activity of 1-5 micromolar required). Thus, whilst each component of the screening system appears to be working individually (a gel system can be constructed in which peptides are synthesised from bead-immobilised DNA templates in-gel, and bacterial cells will grow as a monolayer at the interface of a 2-gel system), final optimisation of integrated performance cannot be performed. To circumvent this, the gel-display screening system will be adapted for optimisation using peptides that prevent antibiotic resistance rather than causing bacterial cell death per se. In this way, potency can be tuned by altering the concentration of antibiotic in the culture gel, and the identification of a peptide that inhibits antibiotic resistance will provide a secondary outcome from this project of significant scientific interest.
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| 今後の研究の推進方策 |
In order to validate the gel-display screening system, a peptide that inhibits antibiotic resistance will be identified. This will be achieved through mRNA-display based screening as originally proposed for the second year of this project, but using a beta-lactamase (VIM2) as a target. To this end and collaboration has been initiated with the Christopher Schofield laboratory at the University of Oxford, who are experts in the field of beta-lactamases, and preparation of the target protein is already underway. It is anticipated that the identification of a peptide inhibitor of VIM2 will take around 2 months to complete. Once such a peptide has been identified, it will be used for validation of the gel-display screening system using cells that express the VIM2 gene in the presence of an appropriate antibiotic, and screening for a novel class of antibiotics will be performed as originally proposed. It is anticipated that the final goals of the project (the construction of a novel gel-display screening system and the identification of a new class of antibiotics) will still be achieved.
Additionally, once a peptide inhibitor of the VIM2 beta-lactamase has been identified, it will be characterised at the University of Oxford leading to a secondary research output from this research.
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| 次年度使用額が生じた理由 |
Expenditure on reagents and consumables for FY2015 was somewhat lower than anticipated, leading to carry over of some funds to FY2016. This was partly due to better than expected research progress over the first few months of the project (meaning that costs associated with optimisation experiments were lower than expected), and partly due to delays incurred during the later stages of FY2015 (which briefly stalled expenditure).
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| 次年度使用額の使用計画 |
The original grant proposal for this project requested a budget of \1,900,000 for chemical reagents in FY2016 in order to cover the much higher cost of reagents for peptide synthesis during this part of the project. However, this amount was ultimately reduced to \1,500,000 during acceptance of the proposal. This shortfall will be covered by the reagent savings made during FY2015, and as such, the extra funds will be put towards the purchase of chemicals and consumables for research purposes.
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