2022 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 / imaging system / gene expression / 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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