iGEM 2020 — Team ROADS_SY

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# Team:ROADS SY/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:ROADS SY/Engineering

# Engineering Success

## Ospt4

We have documented Ospt4 as a new part: [BBa\_K3614888](http://parts.igem.org/Part%3ABBa_K3614888).

Apart from what we plan to do with this gene (to reduce the arsenate accumulation by knocking out the gene), future iGEM teams can also over-express this gene in a plant species (or genetically modify another species with this gene) to increase its ability of absorbing arsenic. Species genetically modified in this way can help clean up arsenic-polluted soil and waterbodies.

![](https://static.igem.org/mediawiki/2020/9/95/T--Roads_SY--mechanism.jpg)

After confirming with the bioinformatic method that OsPT4 is the most important factor of AsV uptake, we will do experiments make sure that OsPT4 was the most important factor in AsV uptake, using the biochemical method. A process that could be simplified as Plant —> Measure —> Record —> Repeat.

## Planting the Rice

![](https://static.igem.org/mediawiki/2020/9/91/T--Roads_SY--rice.jpg)

To speed up the process, we will use hydroponic experiments to grow the rice. (All the reasons and/or process we use certain chemicals or process are stated below, in the “What and Why” section)

First, we surface sterilize the rice seeds for 30 min with diluted Sodium Hypochlorite (NaClO), followed by thorough rinsing for 30 min with deionized water. Seeds were germinated in darkness at 25 °C for 3 days.

Then we transfer the germinated seeds to the hydroponic solution: 1.25 mM NH4NO3, 200 μM KH2PO4, 0.35 mM K2SO4, 1 mM CaCl2, 1 mM MgSO4, 20 μM Fe-EDTA, 20 μM H3BO3, 9 μM MnCl2, 0.32 μM CuSO4, 0.77 μM ZnSO4, and 0.39 μM Na2MoO4. This solution will give the rice plants the nutrients and acidic environment it needs to grow. The hydroponic experiments were conducted in a greenhouse with a 16-h-light (30 °C)/8-h-dark (22 °C) photoperiod and ∼70% relative humidity.

In our hydroponic solution mentioned above, we made some modifications to control the expression of OsPT4, 20 μM AsV with 0 or 300 μM KH2PO4 were used as P-deficient As treatment (−P +As) and P-sufficient As treatment (+P +As), respectively.

## Measurements

![](https://static.igem.org/mediawiki/2020/a/a7/T--Roads_SY--measurement.jpg)

P-Concentrations: ∼0.05 g of crushed dry sample was digested with H2SO4−H2O2 at 300 °C. After cooling, the digested samples were diluted to 100 mL in distilled water. P concentration was analyzed by the molybdenum blue method based on dry weight. The determination of phosphorus, arsenic, silicon and germanium are examples of the use of heteropoly-molybdenum blue in analytical chemistry. The following example describes the determination of phosphorus. A sample containing the phosphate is mixed with an acid solution of MoVI, to produce [PMo12O3]-40. This anion is then reduced to form the blue colored ion, [PMo12O7]-40.The amount of the blue colored ion produced is proportional to the amount of phosphate present and the absorption can be measured using a colorimeter to determine the amount of phosphorus (See diagram above). [3]
As-Concentration: To determine AsV amounts, 17-d old rice plants will be transferred to growth media containing various AsV concentrations (1, 5, 10, 25, and 50 μM) in the absence and presence of 100 mM P. After 30 min, the roots will be rinsed briefly with deionized water, and the shoots and roots separated and dried, which we will ground and digest with HNO3 to determine As concentration using ICP-MS or AFS. However, the advanced machinery used for testing As concentration may even be too hard for us to get, so we may need to find some different ways to test the concentration of AsV, the more common ways are ICP-MS or AFS.

## Gathering Data

Below is the results we expect from our experiments. The data below represents an average number, instead of data from a single experiment. When we are actually conducting our experiments, we will have many groups at the same time, and we omit the highest and lowest concentrations for every group, and plot the averages.

![](https://static.igem.org/mediawiki/2020/b/bb/T--Roads_SY--graph.jpg)

Figures are not drown to scale.

## Conclusion and Further Research

Our research will have a dual impact. It will a) demonstrate that bioinformatics can improve the efficiency and safety of genetic engineering research and b) examine whether the knockout of the (OsPT4) gene could result in a significant reduction in arsenic deposition and contribute to food safety and public health.

After our research on this topic has finished, we can continue experimenting with the other genes in the Pht1 family, like the possible factors of AsV uptake, OsPT1 and OsPT8, and the less studied ones such as OsPT2,3 or 5. This would help lower the AsV uptake even more, lowering the As levels so that the amount of Arsenic in human bodies coming from rice would not harm humans.

### What and Why

1. Sterilizing seeds with NaClO: This removes surface bacteria and fungi without destroying the internal organisms.
2. Ammonium Nitrate (NH4NO3): Using ammonium nitrate in gardens and large scale agricultural fields enhances plant growth and provides a ready supply of nitrogen from which plants can draw. Nitrogen is so vital because it is a major component of chlorophyll, the compound by which plants use sunlight energy to produce sugars from water and carbon dioxide (i.e., photosynthesis). It is also a major component of amino acids, the building blocks of proteins.
3. Potassium Dihydrogen Phosphate (KH2PO4), Potassium Sulfate (K2SO4):This fertilizer is necessary for plants to metabolize Iron (Fe). Potassium is associated with the movement of water, nutrients and carbohydrates in plant tissue. It's involved with enzyme activation within the plant, which affects protein, starch and adenosine triphosphate (ATP) production. The production of ATP can regulate the rate of photosynthesis.
4. Calcium Chloride (CaCl2), Magnesium Sulfate (MgSO4), Manganese (II) Chloride (MnCl2), Copper (II) Sulfate (CuSO4), Zinc (II) Sulfate (ZnSO4), Sodium Molybdate (Na2MoO4): Useful fertilizers for plants, because they provide important micronutrients that are vital to healthy plant growth and development.
5. Fe-EDTA (C10H12FeN2O8): Serves as a very stable plant available iron nutrient.
6. Boric Acid (H3BO3): Boric acid can stop the growth of fungi, such as molds, when added to the solution in small amounts. In addition, boric act can be used to modify the pH value of the rice plant to an ideal value of 6.0~7.0. And because this is an ideal growth environment for rice, it is logical to assume that farmers who grow rice to maintain their rice crops within this acidity, which diminishes the gap between our plants and those sold on market. [1]
7. 70% relative humidity: Nguyen, 2003: “Optimum relative humidity for rice cultivation lies between 60% and 80%”
8. 16-h-light (30 °C)/8-h-dark (22 °C): High temperature especially during night leads to loss of reserved food through greater respiration. For higher grain yield a day temperature of 25 to 32°C and night temperature of 15 to 20°C is preferable. Temperature beyond 35°C affects not only pollen shedding but also grain filling.

## Reference

[1] https://www.uaex.edu/publications/pdf/mp192/chapter-9.pdf

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:ROADS SY/Experiments

# Experiments

## Planting the Rice

![](https://static.igem.org/mediawiki/2020/9/91/T--Roads_SY--rice.jpg)

To speed up the process, we will use hydroponic experiments to grow the rice. (All the reasons and/or process we use certain chemicals or process are stated below, in the “What and Why” section)

First, we surface sterilize the rice seeds for 30 min with diluted Sodium Hypochlorite (NaClO), followed by thorough rinsing for 30 min with deionized water. Seeds were germinated in darkness at 25 °C for 3 days.

Then we transfer the germinated seeds to the hydroponic solution: 1.25 mM NH4NO3, 200 μM KH2PO4, 0.35 mM K2SO4, 1 mM CaCl2, 1 mM MgSO4, 20 μM Fe-EDTA, 20 μM H3BO3, 9 μM MnCl2, 0.32 μM CuSO4, 0.77 μM ZnSO4, and 0.39 μM Na2MoO4. This solution will give the rice plants the nutrients and acidic environment it needs to grow. The hydroponic experiments were conducted in a greenhouse with a 16-h-light (30 °C)/8-h-dark (22 °C) photoperiod and ∼70% relative humidity.

In our hydroponic solution mentioned above, we made some modifications to control the expression of OsPT4, 20 μM AsV with 0 or 300 μM KH2PO4 were used as P-deficient As treatment (−P +As) and P-sufficient As treatment (+P +As), respectively.

## Measurements

![](https://static.igem.org/mediawiki/2020/a/a7/T--Roads_SY--measurement.jpg)

P-Concentrations: ∼0.05 g of crushed dry sample was digested with H2SO4−H2O2 at 300 °C. After cooling, the digested samples were diluted to 100 mL in distilled water. P concentration was analyzed by the molybdenum blue method based on dry weight. The determination of phosphorus, arsenic, silicon and germanium are examples of the use of heteropoly-molybdenum blue in analytical chemistry. The following example describes the determination of phosphorus. A sample containing the phosphate is mixed with an acid solution of MoVI, to produce [PMo12O3]-40. This anion is then reduced to form the blue colored ion, [PMo12O7]-40.The amount of the blue colored ion produced is proportional to the amount of phosphate present and the absorption can be measured using a colorimeter to determine the amount of phosphorus (See diagram above). [3]
As-Concentration: To determine AsV amounts, 17-d old rice plants will be transferred to growth media containing various AsV concentrations (1, 5, 10, 25, and 50 μM) in the absence and presence of 100 mM P. After 30 min, the roots will be rinsed briefly with deionized water, and the shoots and roots separated and dried, which we will ground and digest with HNO3 to determine As concentration using ICP-MS or AFS. However, the advanced machinery used for testing As concentration may even be too hard for us to get, so we may need to find some different ways to test the concentration of AsV, the more common ways are ICP-MS or AFS.

## Gathering Data

Below is the results we expect from our experiments. The data below represents an average number, instead of data from a single experiment. When we are actually conducting our experiments, we will have many groups at the same time, and we omit the highest and lowest concentrations for every group, and plot the averages.

![](https://static.igem.org/mediawiki/2020/b/bb/T--Roads_SY--graph.jpg)

Figures are not drown to scale.

## What and Why

1. Sterilizing seeds with NaClO: This removes surface bacteria and fungi without destroying the internal organisms.
2. Ammonium Nitrate (NH4NO3): Using ammonium nitrate in gardens and large scale agricultural fields enhances plant growth and provides a ready supply of nitrogen from which plants can draw. Nitrogen is so vital because it is a major component of chlorophyll, the compound by which plants use sunlight energy to produce sugars from water and carbon dioxide (i.e., photosynthesis). It is also a major component of amino acids, the building blocks of proteins.
3. Potassium Dihydrogen Phosphate (KH2PO4), Potassium Sulfate (K2SO4):This fertilizer is necessary for plants to metabolize Iron (Fe). Potassium is associated with the movement of water, nutrients and carbohydrates in plant tissue. It's involved with enzyme activation within the plant, which affects protein, starch and adenosine triphosphate (ATP) production. The production of ATP can regulate the rate of photosynthesis.
4. Calcium Chloride (CaCl2), Magnesium Sulfate (MgSO4), Manganese (II) Chloride (MnCl2), Copper (II) Sulfate (CuSO4), Zinc (II) Sulfate (ZnSO4), Sodium Molybdate (Na2MoO4): Useful fertilizers for plants, because they provide important micronutrients that are vital to healthy plant growth and development.
5. Fe-EDTA (C10H12FeN2O8): Serves as a very stable plant available iron nutrient.
6. Boric Acid (H3BO3): Boric acid can stop the growth of fungi, such as molds, when added to the solution in small amounts. In addition, boric act can be used to modify the pH value of the rice plant to an ideal value of 6.0~7.0. And because this is an ideal growth environment for rice, it is logical to assume that farmers who grow rice to maintain their rice crops within this acidity, which diminishes the gap between our plants and those sold on market. [1]
7. 70% relative humidity: Nguyen, 2003: “Optimum relative humidity for rice cultivation lies between 60% and 80%”
8. 16-h-light (30 °C)/8-h-dark (22 °C): High temperature especially during night leads to loss of reserved food through greater respiration. For higher grain yield a day temperature of 25 to 32°C and night temperature of 15 to 20°C is preferable. Temperature beyond 35°C affects not only pollen shedding but also grain filling.

## Reference

[1] https://www.uaex.edu/publications/pdf/mp192/chapter-9.pdf

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:ROADS SY/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)