2021 Fiscal Year Research-status Report
Spatiotemporal mapping of gene expression to reveal mechanisms of cellular differentiation in E. coli biofilms
| Project/Area Number |
21K05341
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| Research Institution | Kyoto University |
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Principal Investigator |
Robert Martin 京都大学, 薬学研究科, 特定准教授 (90365487)
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | biofilm / E. coli / cell motility / surface properties / nutrients / differentiation / gene expression |
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| Outline of Annual Research Achievements |
We focused our efforts on finding the conditions to generate reproducible biofilms with the expected structural complexity. This is expected to be important in providing an environment suitable for cellular differentiation and division of labor, one of the important focus of our study. By manipulating both the growth surface properties (agar hardness) and the nutritional conditions (salt-free agar with various concentrations of glucose) we produced biofilms having significant size and structural diversity. We also monitored the activity of several E. coli promoters using GFP reporter strains under these conditions. We observed patterns of promoter activity (gene expression) that varied widely depending on promoter and also as expected displayed some variation in spatial expression pattern.
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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
The research is progressing as expected and we have been able to test multiple growth conditions and different promoters and E. coli strains. We are now ready the start the proteome analysis and continue the promoter activity analysis on a larger scale as originally planned.
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| Strategy for Future Research Activity |
We will continue to produce biofilms expressing different promoter reporters to monitor gene expression in space and time during development. This will be complemented with proteome analysis during the second year.
For gene expression analysis, in addition to the the E. coli fluorescent promoter collection we will also test and use a different set of YFP-fusion protein strains where gene expression can be directly visualized at the protein level.
To increase experimental throughput we will need extra sets of imaging systems, some of which are no longer commercially available. We are now planning to build some ourselves using open source resources and custom built and 3D-printed components.
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| Causes of Carryover |
Just at the beginning of the project the PI unexpectedly received an incubator from another researcher (transfer) delaying the need for the original purpose. When needed at the end of the fiscal year because of expansion of project scale, it was stock limitation and delivery delays made it impossible to secure in the first fiscal year. Also due to Covid-19 limitations, no business travel expenses were used. The unused money will be used early in the next fiscal year to purchase the incubator and other experimental resources and for travel if the conditions make it possible.
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Research Products
(1 results)
