iGEM 2022 — Team anatolia-college-hs

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Our filter was created only using 3D-printed parts {% endblock %}
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#### Our Design

While creating the filter, we aimed to make it usable and easy to build. The filter consists of the main body and two removable filter nets. The main body is cylindrical with a diameter of 10 centimeters and can be glued to any PVC pipe using Bushing Fittings. The cylinder is dissected into two parts where two specifically designed filter nets are placed. The first filter net keeps bigger particles, such as heavy metals, and the second one prevents the enzymes from escaping. The filter nets can be easily removed from the main body to be cleaned or replaced. To develop the filter, we used the CAM software Fusion 360 and printed it using a 3D printer.

##### Take a look at our main filter design, along with its constituents below!

![](https://static.igem.wiki/teams/4503/wiki/screenshot-2022-10-10-at-11-36-20-pm.png)

Design of the main body of our filter, with the two slots for the insertion of the filter membranes clearly visible.

![](https://static.igem.wiki/teams/4503/wiki/screenshot-2022-10-10-at-11-40-38-pm.png)

Constituent "rings" of the main body of out filter. The curved part allows for easy removal by hand, while the protruding elements ensure that the filter membranes will be held together securely.

##### Manufacture

The design of our filter was optimized mathematically to use as less plastic as possible, while not compromising integral structural strength and stability. It is a scaled down version of the filter that would actually be applied in real life, taking into account the size of water drainage systems in our city. Two prototypes have been produced, adding structural support by making the "rings" wider in diameter and more compartments to slow the flow of the water, and therefore lower the pressure exerted on the filter membranes.

##### Check out the 3D printing process of our filter!

![](https://static.igem.wiki/teams/4503/wiki/filter.jpg)

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{% block title %}Experiments{% endblock %}
{% block lead %}Read more about our experiment and protocols!{% endblock %}
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## Experimental Procedure

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1. Amplification of nitrate reductase gene (nirS) from Pseudomonas azotoformans DNA
DNA of Pseudomonas azotoformans (DSM 18862), strain LMG21611 isolated from paddies in Japan, was purchased from DSMZ Leibniz Institute (Germany). The nirS gene was amplified using PCR. The primers used were based on the NCBI reference sequence: NZ\_LT629702.1 and were designed using Primer-3 software (Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M and Rozen SG.
Primer3--new capabilities and interfaces.
Nucleic Acids Res. 2012 Aug 1;40(15):e115.) In order to make directional cloning possible, the sites of two restriction enzymes were incorporated into the Primer sequence. The resulting Primers are: NirS-F 5' GTCAATCATATGCCCCCGACGGCCCAT3'
NirS-R 5'TGGTGGTTCGAATCAATAAATATCGTGTTGGG3'. In the forward Primer the sequence for NDEI and at the reverse the HindIII restriction site sequence. The PCR reaction was performed using fusion high fidelity DNA polymerase master mix (Thermo scientific). The reaction mixture contained the following: 25 μl Fusion master mix, 5 μl DNA, 1.5 μl nirS-F Primer (10 mM), 1.5 μl nirS-R Primer (10 mM), and 17 μl PCR-grade water. The PCR program used was: 95o C for 5 minutes, followed by 35 cycles of denaturation at 95o C for 30 seconds, annealing 60o C for 1 minute, extension 72o C for 1.5 minute. A final extension step was performed at 72o C for 5 minutes. The PCR products were visualized on an 1% agarose gel in TAE buffer. The expected size of the amplicon was 1626 bp.

## Purification of nirS fragment.

The gel band that appeared at 1626 bp was excised and the DNA fragment was purified using the MACHEREY-NAGEL PCR NucleoSpin Gel and PCR Clean‑up, following manufacturers’ instructions. The DNA fragment was eluded in 30 μl of warm ellusion buffer.
Transformation of E. coli with pET 29c + vector.

## Plasmid mini-prep

Colonies that grew on LB+kanamycin plates (30 μg per mL)

## Transformation of E. coli with pET 29c + vector

E. coli competent cells and the pET29c+ vector were a gift from the laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki.

- Equilibrate a dry bath to 42°C.
- Equilibrate a dry bath to 42°C.
- Add 1 μL of plasmid DNA (10 pg to 100 ng) to a vial of thawed competent cells.
- Incubate on ice for 30 minutes.
- Heat shock cells for 30 seconds at 42°C.
- Place on ice for 2 minutes.
- Add 950 microliters of SOC medium to the vial.
- Incubate tube in a 37°C water bath for 1 hour. Because a shaking incubator was not available, one of the team members was shaking the vial manually.
- Spread from 50 - 200 microliters on LB + 30 μg/mL kanamycin selective plates. The plates were pre-warmed to 37°C, Incubate overnight at 37°C.

## Plasmid mini-prep

- Colonies that grew on LB+kanamycin plates were picked using a sterile pipette tip and placed in a sterile test tube containing 3 mL of LB broth + 30μL/mL kanamycin. The test tubes were incubated overnight at 37°C.
- One mL of growth medium was transferred in a sterile 1.5 mL test tube and centrifuged at 11000 x g for 5 minutes.
- The supernatant was removed and the pellet was resuspended in 250 μL Buffer A1 by pipetting up and down and vortexing until no visible clumps could be detected. The NucleoSpin Plasmid, Mini kit for plasmid DNA (Macherey Nagel) was used following manufacturer’s instructions.
- The presence of the plasmid was verified by gel electrophoresis using a 1% agarose gel in TAE buffer. The plasmid miniprep was kept at -20°C until use.

## Inspirations

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* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

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

## Describe what your results mean

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* 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

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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.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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