Spatiotemporal mapping of gene expression to reveal mechanisms of cellular differentiation in E. coli biofilms
Project/Area Number |
21K05341
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Research Category |
Grant-in-Aid for Scientific Research (C)
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Allocation Type | Multi-year Fund |
Section | 一般 |
Review Section |
Basic Section 38020:Applied microbiology-related
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Research Institution | Kyoto University |
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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Project Status |
Granted (Fiscal Year 2022)
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Budget Amount *help |
¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2023: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
Fiscal Year 2022: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2021: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
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Keywords | biofilm / E. coli / imaging system / gene expression / proteome / cell motility / surface properties / nutrients / differentiation / Escherichia coli |
Outline of Research at the Start |
Bacteria can form biofilms having unique and emergent biological properties. To explore these properties we will quantify gene expression and metabolic activity in E. coli biofilms using time-lapse fluorescence imaging of gene expression, and spatio-temporal mapping of the cellular proteome.
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Outline of Annual Research Achievements |
During the second fiscal year we have progressed on multiple fronts. 1) We started and progressed with spatial analysis of protein expression by sampling biofilms at different radial locations followed by ongoing proteome analysis. 2) We characterized further expression of selected promoters activity under different conditions. To normalize the fluorescence results we have been testing a secondary fluorescent protein whose expression is constitutive. 3) We have been characterizing the effect of glucose addition on biofilm development and our results suggest that an unsuspected metabolic imbalance may be important in the process. 4) We have developed imaging systems based on Raspberry Pi computers and cameras to collect time-lapse images in dual fluorescence mode in addition to bright field.
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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 project has moved forward very well along different and complementary directions. Most of the originally proposed methods are working well and we are able to collect data smoothly. Different components of the research project are progressing in parallel and producing promising results.
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Strategy for Future Research Activity |
Because cell density is not uniform across biofilms, there are some challenges with normalizing signals. We are working on resolving this using a secondary fluorescent protein as a reporter of cells density. We will also analyze in more details the systems dynamics by quantifying biofilm growth during development. These experiments can provide information on environmental changes during growth and complement the gene expression analysis.
In the third year, we will continue the proteome analysis in more details in parallel with promoter activity and fusion protein expression. To analyze the complexity of gene expression in 3D we also plan to try confocal microscopy as well as imaging of thin slices of biofilms.
Further improvements to our automated multi-mode imaging system will be made.
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Report
(2 results)
Research Products
(3 results)