2017 Fiscal Year Research-status Report
Visualization of metabolic dynamics during pattern formation in bacteria
| Project/Area Number |
16K07333
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| Research Institution | Tohoku University |
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Principal Investigator |
Robert Martin 東北大学, 高度教養教育・学生支援機構, 准教授 (90365487)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | bacterial macro-colonies / gene expression / fluorescence / biofilm / c-di-GMP |
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| Outline of Annual Research Achievements |
Efforts focused on developing, and using imaging systems for capturing time-lapse video of growing macro-colonies in both bright field and fluorescence mode. Both direct photography using an dSLR camera as well as microscopic systems equipped with digital cameras were set up and tested.
Various Venus-GFP fusion strains were selected representing various metabolic processes (enzymes) or regulators (like Crp and RpoS). We have been able to observe strong Venus-GFP expression of some of the selected genes under various nutritional and agar hardness conditions. For some genes, expression within the pattern appear weak does not seem to vary much and we will now investigate this more in the third dimension, from the top to the bottom of the colony.
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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
Although delayed at the beginning of the fiscal year more intensive efforts were placed in the latter part and to develop the experimental setup for visualization. Experiments are proceeding proceeding smoothly.
Some imaging systems have been setup and still images of multiple colonies (as well as some time-lapse movies) growing under different conditions have been collected. The results obtained already point to significant differences between the strains and nutritional conditions.
A student has also been trained and will continue to support the project efforts in the third year to increase the pace of the experimental flow.
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| Strategy for Future Research Activity |
The project is moving forward well and various types of colony patterns have been produced and analyzed for gene expression using both still and time-lapse video imaging. The next step will be to expand such visualization efforts to a larger number of Venus GFP-fusion strains (multiple metabolic enzymes) under various experimental conditions.
As mentioned above, when little variation in gene expression is observed from observation of the whole colony, we will slice the colony vertically through the agar using a cryostat using published methods to obtain a transversal view of gene expression (from top to bottom of the colony). In addition, we plan to use such slices to perform metabolite analysis in and around the colony using MS-imaging as originally proposed.
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| Causes of Carryover |
The scanner and computer were not purchased yet as other forms of imaging were considered as priority since the scanner cannot be used for fluorescence, the main focus of the gene expression part of the project. Still we are considering modifying the scanner for fluorescence use as it would contribute to increase the throughput of the analysis of macrocolonies.
Unused funds from the second year will be used to purchase of a computer need to control some imaging systems and research consumables. Funds from the unused travel budget might be used to invite an expert to help for some imaging.
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Research Products
(2 results)
