iGEM 2021 — Team Nanjing-China

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# Team:Nanjing-China/Notebook

Nanjing-China/Notebook

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[SUSTAINABLE DEVELOPMENT](https://2021.igem.org/Team%3ANanjing-China/Sustainable)

[ACHIEVEMENTS](https://2021.igem.org/Team%3ANanjing-China/Achievements)

SOCIAL PRACTICES

[Human practices](https://2021.igem.org/Team%3ANanjing-China/Human_Practices)

[Education & Communication](https://2021.igem.org/Team%3ANanjing-China/Education)

[Collaborations](https://2021.igem.org/Team%3ANanjing-China/Collaborations)

[Partnership](https://2021.igem.org/Team%3ANanjing-China/Partnership)

[Inclusivity](https://2021.igem.org/Team%3ANanjing-China/Inclusivity)

[PARTS](https://2021.igem.org/Team%3ANanjing-China/Parts)

PROJECT

[Description](https://2021.igem.org/Team%3ANanjing-China/Description)

[Design](https://2021.igem.org/Team%3ANanjing-China/Design)

[Engineering](https://2021.igem.org/Team%3ANanjing-China/Engineering)

[Experiments](https://2021.igem.org/Team%3ANanjing-China/Experiments)

[Model](https://2021.igem.org/Team%3ANanjing-China/Model)

[Safety](https://2021.igem.org/Team%3ANanjing-China/Safety)

[Notebook](https://2021.igem.org/Team%3ANanjing-China/Notebook)

[Implementation](https://2021.igem.org/Team%3ANanjing-China/Implementation)

[Contribution](https://2021.igem.org/Team%3ANanjing-China/Contribution)

TEAM

[Members](https://2021.igem.org/Team%3ANanjing-China/Members)

[Attributions](https://2021.igem.org/Team%3ANanjing-China/Attributions)

[HOME](https://2021.igem.org/Team%3ANanjing-China)

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PolyP preparation

1. PolyP synthesis in bacteria

(1) Monoclonal streaking inoculation

The engineered bacteria containing the target plasmid
(produced by the company according to the plasmid sequence and stored in a glycerol tube)
were streaked on the Kana plate and cultured overnight at 37°C.

(2) LB culture

1) Prepared a 250ml Erlenmeyer flask, added 50ml LB, 50μl (1000x) Kana.

2) Used a pipette tip to pick the monoclonal strain on the plate into an Erlenmeyer flask and shook it well.
Cultured on shaker overnight at 37°C (12h).

3) Measured the absorbance of the bacterial solution at OD600 with LB zero adjustment,
determined the amount of bacterial solution added to the synthetic wastewater culture medium,
and ensured that the initial OD600 of the synthetic wastewater culture medium containing engineered bacteria was 0.2.

(3) Cultivation of synthetic wastewater

1) Centrifuged the bacteria-containing LB medium at 5000r for 1min, discarded the supernatant,
resuspended it with a small amount of synthetic wastewater medium, pipette and mix well,
centrifuged at 5000r for 1min, discarded the supernatant, and resuspended with a small amount of synthetic wastewater.

2) Prepared a 500ml Erlenmeyer flask, added 200ml synthetic wastewater culture medium, 200μl (1000X) Kana to each bottle,
calculated the amount of synthetic wastewater culture solution after resuspension. Incubated on a shaker at 37°C for 12-14h.

(4) Determination of polyP content

1) Centrifuged at 12000r for 1min to separate the bacteria and the solution.

2) Took the supernatant and used the molybdate method to determine the phosphorus content in the solution.
The reduced phosphorus was considered to be synthesized as polyP and stored in the bacteria.

2.Extraction of polyP

(1) Bacteria pretreatment

The recombinant engineered bacteria (containing polyP) cultured in synthetic wastewater for 14 hours
were freeze-dried to obtain freeze-dried powder, and placed in a muffle furnace to heat at 600 degrees for 1 hour
to obtain a carbonized black solid.

(2) Ground the black solid, added deionized water to dissolve it,
the supernatant solution was the product solution containing polyP,
placed it in a freeze dryer and freeze-dry to obtain the solid.

3. Purification of polyP (separation of polyP with different chain lengths)

(1) Used NaOH or KOH (2.5M) to adjust the pH of the polyP aqueous solution to 7, meanwhile, the total volume of the solution was V1.

(2) In order to precipitate polyP with medium chain length, 0.156V1 volume of ethanol (96%v/v) was added to the solution and mixed well.
The solution was stood for 1 hour to allow the polyP to fully aggregate together.

(3) Centrifuged the solution at 10000g for 5min after standing to separate the insoluble matter from the supernatant.
The insoluble matter contained medium chain polyP (polymerization degree 30-40).

(4) Desiccation. Opened the cap of the above centrifuged tube and placed it on a desiccator (a device filled with silica) for one week.

(5) Separated short-chain polyP, and collected the supernatant in step (3) into a new tube.
At this time, the supernatant contained short-chain polyP (polymerization degree is about 11).

(6) Added 0.885V1 volume of absolute ethanol and mixed well.
The solution was allowed to stand for 1 hour to allow the polyP to fully aggregate together.

(7) Centrifuged the solution at 10000g for 10min after standing, the insoluble matter contained short-chain polyP.

(8) Desiccation, same as step (4).

4. Determination of phosphorus content in solution by molybdate method

(1) Configuration of vitamin C and molybdate solution

1) Prepared 10g/100ml vitamin C solution and stored it in a brown bottle.

2) Dissolved 13g of ammonium molybdate tetrahydrate in 100ml of water and slowly added it to 300ml of 49% sulfuric acid.
Dissolved 0.35g potassium antimony tartrate into 100ml water, slowly added and mixed well, stirring while adding. Stored in a brown bottle.

(2) Used 10ml molecular water+200μl vitamin C solution + 400μl molybdate solution to zero,
set 9.8ml molecular water + 200μl synthetic wastewater + 200μl vitamin C solution + 400μl as the total amount,
centrifuged 9.8ml molecular water + 200μl Clear liquid + 200μl vitamin C solution + 400μl as the experimental group,
the value of OD700 was measured under an ultraviolet spectrophotometer.
According to the formula of the remaining amount of phosphorus in the experimental group (unit mg/l) =
(total OD700 - experimental group OD700) \* 100, calculated the polyphosphate content in the bacteria.

5. Determination of the number of bacteria

(1) Used pure mediums(LB or PA) to zero, the mediums containing bacterial as experimental group,
the value of OD600 was measured under an ultraviolet spectrophotometer.

(2) If OD600 is larger than 1.00, we thought that the results were beyond the detection limit at one single test,
so we need to do 6 times dilution and detect again. The testing results need to time 6.

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# Team:Nanjing-China/Engineering

Nanjing-China/Engineering

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[SUSTAINABLE DEVELOPMENT](https://2021.igem.org/Team%3ANanjing-China/Sustainable)

[ACHIEVEMENTS](https://2021.igem.org/Team%3ANanjing-China/Achievements)

SOCIAL PRACTICES

[Human practices](https://2021.igem.org/Team%3ANanjing-China/Human_Practices)

[Education & Communication](https://2021.igem.org/Team%3ANanjing-China/Education)

[Collaborations](https://2021.igem.org/Team%3ANanjing-China/Collaborations)

[Partnership](https://2021.igem.org/Team%3ANanjing-China/Partnership)

[Inclusivity](https://2021.igem.org/Team%3ANanjing-China/Inclusivity)

[PARTS](https://2021.igem.org/Team%3ANanjing-China/Parts)

PROJECT

[Description](https://2021.igem.org/Team%3ANanjing-China/Description)

[Design](https://2021.igem.org/Team%3ANanjing-China/Design)

[Engineering](https://2021.igem.org/Team%3ANanjing-China/Engineering)

[Experiments](https://2021.igem.org/Team%3ANanjing-China/Experiments)

[Model](https://2021.igem.org/Team%3ANanjing-China/Model)

[Safety](https://2021.igem.org/Team%3ANanjing-China/Safety)

[Notebook](https://2021.igem.org/Team%3ANanjing-China/Notebook)

[Implementation](https://2021.igem.org/Team%3ANanjing-China/Implementation)

[Contribution](https://2021.igem.org/Team%3ANanjing-China/Contribution)

TEAM

[Members](https://2021.igem.org/Team%3ANanjing-China/Members)

[Attributions](https://2021.igem.org/Team%3ANanjing-China/Attributions)

[HOME](https://2021.igem.org/Team%3ANanjing-China)

![picture display fail](https://static.igem.org/mediawiki/2021/9/92/T--Nanjing-China--NEW_ENGINEERING.png "cover picture")

[Design](#part1)

[Build&Test](#part2)

[Learn](#part3)

[Redesign&Rebuild](#part4)

[More information](#part5)

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# Team:Nanjing-China/Experiments

Nanjing-China/Experiment

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[![picture display fail](https://static.igem.org/mediawiki/2021/f/f7/T--Nanjing-China--teamlogo_white.png "team logo")](https://2021.igem.org/Team%3ANanjing-China)

[SUSTAINABLE DEVELOPMENT](https://2021.igem.org/Team%3ANanjing-China/Sustainable)

[ACHIEVEMENTS](https://2021.igem.org/Team%3ANanjing-China/Achievements)

SOCIAL PRACTICES

[Human practices](https://2021.igem.org/Team%3ANanjing-China/Human_Practices)

[Education & Communication](https://2021.igem.org/Team%3ANanjing-China/Education)

[Collaborations](https://2021.igem.org/Team%3ANanjing-China/Collaborations)

[Partnership](https://2021.igem.org/Team%3ANanjing-China/Partnership)

[Inclusivity](https://2021.igem.org/Team%3ANanjing-China/Inclusivity)

[PARTS](https://2021.igem.org/Team%3ANanjing-China/Parts)

PROJECT

[Description](https://2021.igem.org/Team%3ANanjing-China/Description)

[Design](https://2021.igem.org/Team%3ANanjing-China/Design)

[Engineering](https://2021.igem.org/Team%3ANanjing-China/Engineering)

[Experiments](https://2021.igem.org/Team%3ANanjing-China/Experiments)

[Model](https://2021.igem.org/Team%3ANanjing-China/Model)

[Safety](https://2021.igem.org/Team%3ANanjing-China/Safety)

[Notebook](https://2021.igem.org/Team%3ANanjing-China/Notebook)

[Implementation](https://2021.igem.org/Team%3ANanjing-China/Implementation)

[Contribution](https://2021.igem.org/Team%3ANanjing-China/Contribution)

TEAM

[Members](https://2021.igem.org/Team%3ANanjing-China/Members)

[Attributions](https://2021.igem.org/Team%3ANanjing-China/Attributions)

[HOME](https://2021.igem.org/Team%3ANanjing-China)

![picture display fail](https://static.igem.org/mediawiki/2021/1/1d/T--Nanjing-China--NEW_EXPERIMENTS.png "cover picture")

[Background](#part1)

[Purpose](#part2)

[Process](#part3)

[Experimental results](#part4)

[Conclusion](#part5)

[Attachments](#part6)

Experiment in vitro

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Nanjing-China/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.

* [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)