2016 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 colonies / pattern formation / bacterial strains / c-di-GMP / biofilm |
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| Outline of Annual Research Achievements |
Efforts were placed on preparing various E. coli strains necessary for the project. Following this, various culture conditions were tested and optimized to produce different colony patterns. These included multiple nutritional, agar concentration (hardness) as well as temperature and humidity conditions. These important but preliminary experiments have allowed to produce some of the expected patterns such as eden-like and DLA-type patterns but also biofilm-like wrinkled colonies that formed interesting and different structures depending on agar concentrations. After extended periods of colony growth, some cellular projections within the agar plate (corona pattern) was observed in some cases.
Tests to evaluate possible effects of c-di-G(A)MP analogs on pattern formation have also been performed. One issue at first has been that the analogs are relatively insoluble in aqueous solutions used for culture. Different solvents were tested and different application methods on agar were tested. Biofilm structures were generated in absence or presence of increasing amounts of analogs. Preliminary tests show that c-di-GMP might interfere with pattern formation. Some effect was observed at high analog concentration and especially visible for longer incubation time. However, these results needs to be confirmed by repeating the experiments further.
Overall the results confirm that we are able to reproduce and create original patterns that are reproducible and performed preliminary experiments with nucleotide analogs.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
There has been some delay in the execution of some of the planned experiments because it has taken longer than expected to produce some patterns and testing new ones. Finding and optimizing environmental conditions for keeping plates in good conditions and limit drying of plates during long culture times limited progress to some extent. Some interesting patterns take a long time to produce, also limiting the total number of experiments that could be performed.
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| Strategy for Future Research Activity |
The project is still at an early stage but interesting patterns have now been produced reproducibly using wild-type or control E. coli strains. We will therefore now focus efforts on the most promising ones to begin visualizing the metabolic gene expression within those patterns. For this we plan to proceed according to the original plan using selected strains of E. coli bearing Venus GFP-fused to metabolic genes and observe patterns of expression in a spatiotemporal way.
The first preliminary tests of pattern formation using wild-type E. coli reveal that some di-nucleotide analogs may have some effects on pattern formation but this remains to be confirmed. We will use both commercially available c-di-GMP and the synthetic analogs to probe this process further and confirm the possible effects observed in preliminary experiments during the first year.
As time permits we will also begin to analyze metabolite production in and around colony structures to connect the formation process to specific biochemical messengers.
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| Causes of Carryover |
As mentioned above, due to slower than expected progress with some of the experiments in the first year, some of the planned equipment and consumable purchases have not yet been made. In addition, due to commitments of the PI and schedule constrains no funds were used for traveling overseas in 2016.
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| Expenditure Plan for Carryover Budget |
The funds from the first year that remain unused will be used mostly as planned in the second year. This includes the purchase of a computer and some scanner(s) for the real-time monitoring of colony growth and research consumables.
The unused part for personnel remuneration that was not used in 2016 will be combined with the part for 2017 to try to allow for longer or multiple opportunities technical support. Funds from the unused travel budget might be used to invite some researcher for collaboration.
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Research Products
(2 results)
