iGEM 2021 — Team XHD-Wuhan-A-China

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# Team:XHD-Wuhan-A-China/Notebook

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Notebook

[Notebook](https://static.igem.org/mediawiki/2021/d/de/T--XHD-Wuhan-A-China--Notebook.pdf)

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Copyright ©2021 iGEM XHD-Wuhan-A-China All rights reserved

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# Team:XHD-Wuhan-A-China/Engineering

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Engineering

In order to enhance the denitrification of rhizobia, we designed an enhanced denitrification system.

Enhanced denitrification system

We designed an enhanced denitrification system, which includes two plasmids: napA-pBBR1MCS2 and nirK-pBBR1MCS2. We designed the napA gene circuit J23100-RBS-napA (Figure 1). In order to realize the function of this gene circuit, we assembled this circuit on the pBBR1MCS2 plasmid backbone and connected to amilCP for characterization (Figure 2). At the same time, we designed the nirK gene circuit J23100-RBS-nirK (Figure 3). In order to realize the function of this gene circuit, we assembled this circuit on the pBBR1MCS2 plasmid backbone and connected to amilCP for characterization (Figure 4).

![](https://static.igem.org/mediawiki/2021/2/2e/T--XHD-Wuhan-A-China--Engineering1.png)

Figure 1. Constitution of J23100-RBS-napA gene circuits

![](https://static.igem.org/mediawiki/2021/4/41/T--XHD-Wuhan-A-China--Engineering2.png)

Figure 2. Design of Enhanced denitrification system plasmid (napA-pBBR1MCS2)

![](https://static.igem.org/mediawiki/2021/c/c8/T--XHD-Wuhan-A-China--Engineering3.png)

Figure 3. Constitution of J23100-RBS-nirK gene circuits

![](https://static.igem.org/mediawiki/2021/9/96/T--XHD-Wuhan-A-China--Engineering4.png)

Figure 4. Design of Enhanced denitrification system plasmid (nirK-pBBR1MCS2)

Usage and Biology

We verified the length of the recombinant plasmid by restriction enzyme digestion to ensure the success of the recombinant plasmid (Figure 5). Theoretically, the transformation of napA-pBBR1MCS2 or nirK-pBBR1MCS2 will increase the copy number of napA and nirK genes, increase the content of denitrification enzymes, and improve the denitrification ability of the strain.

We cultivated strains containing napA-pBBR1MCS2, nirK-pBBR1MCS2 and wild-type strain in LB liquid medium. And the content of amilCP was measured by OD588 every 30 minutes, which results are shown in (Figure 6).

![](https://static.igem.org/mediawiki/2021/1/13/T--XHD-Wuhan-A-China--Engineering5.png)

Figure 5. Gel electrophoresis of plasmid MCS-napA(nirk)-amilcp

![](https://static.igem.org/mediawiki/2021/5/5c/T--XHD-Wuhan-A-China--Engineering6_2.png)

Figure 6. Changes in amliCP concentration of LB liquid medium

We have successfully constructed an enhanced denitrification system and successfully verified the reliability of the system to decompose nitrates. Through our system, we increased the number of copies of napA and nirK successfully. We verified the system that expresses napA-anilCP and nirK-anilCP fusion protein over time, so that the optimal culture time can be determined. Our project can provide new solutions for decomposing excessive nitrate in the soil and improving soil compaction.

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# Team:XHD-Wuhan-A-China/Experiments

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Protocols and Experiments

Bacterial genome extration

The experimental steps as follow:

1) The bacteria were cultured at the optimum growth temperature (4 mL) to the logarithmic stage, centrifuged at 12000 rpm for 1 min, and the supernatant was discarded.

2) Add 450 uL sterile water for re-suspension of bacteria solution.

3) Add 50 uL 10% SDS + 5 uL protease K (20 mg/mL) and incubate at 58℃for 2 h.

4) Add 50 uL 5 M NaCl (the final concentration is about 0.5 M).

5) 550 uL phenol/chloroform was added, and fully mixed, centrifuged at 12000 rpm for 10 min.

6) Put the supernatant (450 uL) into a new 1.5 mL centrifuge tube, add equal volume of chloroform (450 uL), mix upside down, centrifuge at 12000 rpm for 10 min.

7) Drain the supernatant (300 uL) into a 1.5 mL centrifuge tube, add 0.1V NaAc (30 uL) and 0.6V isopropyl alcohol (180 uL), mix them upside down gently. Precipitation at -20 ℃ for 30 min. Centrifugation at 12000 rpm for 10 min.

8) The precipitate was washed with 500 uL 75% ethanol and centrifuged at 12000 rpm for 5 min.

9) Dissolve with 50 uL sterile water or TE.

10) DNA purity and concentration were detected by agarose gel electrophoresis.

PCR Process (one cycle)

Overview

1. Predenaturing: 98℃continues 3mins——Strands Separate

2. Denaturing: 98℃ continues 10s——Strands Separate

3. Annealing: 55℃ continues 10s——primers bind template.

4. Extnsion: 72℃ continues 25s——synthesise new strand

5. Go to step2×30

6. Extnsion: 72℃ continues 2 min

7. 4℃ HOLDING

If it is linear plasmid:

98℃ 5 min，98℃ 10 s，65℃15 s，72℃ 4 min，30 cycles，72℃ 5 min，10℃~

If it is gene:

98℃ 5 min，98℃ 10 s，68℃15 s，72℃ 2 min，30 cycles，72℃ 5min，10℃~

Then DpnI digested the annular plasmid (DpnI 1ul, Green buffer 3.5ul) at 37℃ for 5min, 4℃~

Gel Extraction

1. Cut the single target fragment from the agarose gel, place it in a clean centrifuge tube and weigh it.

2. Add three times the volume of PE solution to the gel block. If the gel weight is 0.1g, the volume is considered as 100 uL, and add 300 uL of PE solution. Using a gel cutter to cut 1% agarose gel, the weight of a single piece is about 0.06g. The actual weight of the gel is related to the gel concentration and thickness. Solizing at 15-25℃ for 5-10min at room temperature, during which the centrifugal tube is constantly gently turned up and down to ensure that the gel block is fully dissolved, if the gel block is too large, you can cut the gel block in advance.

3. Add the solution obtained in the previous step into an column CA5 (the adsorption column is placed in the collection tube), place at room temperature for 2 min, centrifuge at 12000 rpm for 30-60 s, pour away the waste liquid in the collection tube, and place the column CA5 into the collection tube.

4. Add 600 uL bleaching solution PW to column CA5 (add anhydrous ethanol before use) centrifuge at 12000 rpm for 30-60s, pour away the waste liquid in the collection tube, and put column CA5 into the collection tube.

5. Repeat the preceding operations.

6. Put the column CA5 back into the collection tube and centrifuge it at 12000 rpm for 2 min to remove the rinse solution as far as possible. The column CA5 was placed at room temperature for several minutes and dried thoroughly to prevent residual bleaching liquid from affecting the next experiment.

7. Put column CA5 into a clean centrifugal tube, add an appropriate amount of eluting buffer TB to the middle of the adsorption film, and place at room temperature for 2 min. DNA solution was collected by centrifugation at 12000 rpm for 2 min.

Plasmid construction

Plasmid construction will be carried out in the afternoon (Ice maker will be turned on at noon).

MCS2 linear Plasmid (4762 bp), required mass 0.02X4762=95.24 ng

NapA gene (2505 bp), required mass 0.04x2505 =100.2 ng

|  |  |
| --- | --- |
| Name | Volume |
| 5 x CE II Buffer | 4μL |
| Exnase II | 2μL |
| MCS2 linear Plasmid | 0.5μL |
| napA gene | 2μL |
| H2O | 11.5μL |

Total 20 uL then incubated with the 37 ℃, 4℃ Holding.

Plasmid transformation

1. Competent cells (e.g. DH5α Competent cell, Vazyme #C502) are thawed on ice.

2. Add 10 μL recombinant product to 100 μL competent cells, lightly flip the tube wall and mix well (do not shake the mixture), then stand on ice for 30 min.

3. After 42℃ water bath heat shock for 45 s, immediately placed on ice to cool for 2-3 min.

4. Add 900 μL SOC or LB medium (without antibiotics), shake bacteria at 37℃ for 1 h (rotation speed 200-250 rpm).

5. The LB solid medium plate with corresponding resistance was preheated in a 37℃ incubator.

6. Centrifugation was performed at 5,000 rpm for 5 min, and 900 μL supernatant was discarded. Hang the bacteria in the remaining medium and lightly coat them on a plate containing the correct resistance using a sterile coating stick.

7. Culture upside down in 37℃ incubator for 12-16 h.

Preparation of Competent cells

(1) Cell was inoculated in LB liquid medium at a volume ratio of 1:100 and cultured overnight at 28℃ until OD600 =0.5-0.6.

(2) Precool the bacteria solution on ice for 10 min. 3 mL bacterial solution was placed in a 1.5 mL sterile centrifuge tube, centrifuged at 4℃ for 5000 r/min for 5 min, and the supernatant was discarded.

(3) Add 1 mL pre-cooled 0.1 mol/L CaCl2 and gently blow and wash the suspended bacteria. Centrifugation was performed at 5000 r/min at 4℃ for 5 min. Supernatant was discarded.

(4) Add 100 μL precooled glycerol CaCl2 to resuspended precipitate and store at -70℃.

Combined transfer

(1) Prepare the membrane and sterilize it in a glass petri dish lined with filter paper. Prepare forceps, sterile water. TY medium, no resistance. Two tablets per medium.

(2) 8ml bacterial solution was centrifuged at 3500g for 5min, and thalli were collected in large quantities.

(3) Then add sterile water twice, then mix 1:1 and wash again, remove supernatant.

(4) Drop the mixed bacterial liquid on the filter membrane, each filter membrane 100 uL, culture for 48 hours, seal the petri dish with the sealing membrane.

(5) The bacteria are then washed off the membrane, into the tube.

(6) After 48 hours, dilute the cells into the plate.

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# Team:XHD-Wuhan-A-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)