2021 Fiscal Year Research-status Report
Investigating spider silk self-assembly via liquid-liquid phase separation
| Project/Area Number |
21K06043
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
Malay Ali 国立研究開発法人理化学研究所, 環境資源科学研究センター, 上級研究員 (40467006)
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | silk / biomimetics / phase separation / spider / nanofibril / self-assembly / biopolymer |
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| Outline of Annual Research Achievements |
As outlined in the project proposal, I have designed and obtained recombinant genetic constructs for expression of spidroins in bacterial, with different domain and/or specific sequence combinations. Heterologous expression in E. coli was carried out, with highly promising results in the case of the MaSp2 spidroin constructs. For MaSp1, low levels of expression necessitated testing different fusion protein platforms for improved yields. Various initial experiments have been carried put to characterize the purified spidroin variants, including generation of phase separation maps, microscopy to probe mesoscale structure self-assembly , Raman spectroscopy, synchrotron small-angle X-ray scattering (SAXS), among others.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
So far, the project is progressing smoothly, and as planned. As mentioned above, in the case of the MaSp1 constructs, inherent issues with the protein solubility and overall yield might require further testing of different expression platforms using empirical methods.
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| Strategy for Future Research Activity |
From the second year of the project, I will focus on the self-assembly mechanisms and structures of the different spidroin constructs. Aside from experiments outlined in the original proposal, depending on the progress of the research, we may also explore alternative experimental approaches, such as super-resolution microscopy, NMR spectroscopy, microfluidics methods for self-assembly, and computational methods for modelling molecular interactions.
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| Causes of Carryover |
There was some delay in the experimental work and therefore the purchasing of some items was delayed. For the next year we will purchase consumable items that will make use of the extra funds.
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