iGEM 2023 — Team sustech-med

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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 %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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## Engineering

### 1. Phage Engineering

#### Overview

Following an exhaustive review of the literature, we initially targeted four genes, wspF ,
yhjH , aiiA , and ytnP , for editing. Employing the introduction of high-copy-number plasmids
for indirect validation, coupled with the elimination of the pf4 sequence for verification,
we demonstrated the feasibility of all four genes as biofilm repressing elements. However,
due to the high failure rate of homologous recombination, successful integration into the
genome was achieved only for aiiA and yhjH .

![4](https://static.igem.wiki/teams/4747/wiki/3-wet-lab/3-3-results/01.png)

#### 1、Electroporation-mediated Transfer of wspF , yhjH , aiiA , and ytnP Gene into pHERD20T Plasmid of P. aeruginosa PAO1

##### a) Preparation of Competent Cells:

* Grow a fresh overnight culture of P. aeruginosa PAO1 in LB broth at 37°C with shaking
  at 200 rpm.
* Harvest the cells by centrifugation at 4000 x g for 10 minutes at 4°C.

##### b) Restriction Digestion and Gel Purification:

* Digest the pHERD20T vector and aiiA /yhjH /wspF /ytnP genes PCR product with suitable
  restriction enzymes.
* Run the digestion products on an agarose gel and extract the desired fragments using a
  gel extraction kit.

##### c) Ligation:

* Ligate the aiiA /yhjH /wspF /ytnP gene fragment into the pHERD20T vector using T4 DNA
  ligase at 16°C overnight.

##### d) Transformation into E. coli :

* Transform the ligation mixture into competent E. coli cells (DH5α) via heat-shock
  method, and select for transformants on LB agar plates.

##### e) Preparation of pHERD20T-aiiA /yhjH /ytnP /wspF Plasmid for Electroporation:

* • Extract the pHERD20T-aiiA /yhjH /ytnP /wspF plasmid from positive E. coli colonies.

##### f) Electroporation into P. aeruginosa PAO1:

* Mix 1-2 µl of the pHERD20T-aiiA /yhjH /ytnP /wspF plasmid with 50 µl of competent P.
  aeruginosa PAO1 cells.
* Transfer the mixture into a pre-chilled electroporation cuvette and electroporate at
  2.5 kV, 200 Ω, 25 µF.
* Immediately add 1 ml of SOC medium and incubate at 37°C for 1 hour with shaking.
* Spread the cells onto LB agar plates supplemented with carbenicillin and incubate at
  37°C overnight.

##### g) Confirmation of electroporation:

We herein report the successful electroporation-mediated transfer of wspF , yhjH , aiiA , and
ytnP genes into the pHERD20T plasmid, as evidenced by PCR analysis (Figure 1).

![4](https://static.igem.wiki/teams/4747/wiki/3-wet-lab/3-3-results/02.png)

August Plan (We have a schedule every month)

![4](https://static.igem.wiki/teams/4747/wiki/3-wet-lab/3-3-results/03.png)

#### 2. Deletion of pf4 Phage Sequence and Plasmid Integration in Pseudomonas aeruginosa PAO1

##### a) Deletion of pf4 Phage Sequence:

Use a SacB system for homologous recombination to facilitate the deletion of the pf4
sequence. Perform PCR to obtain the flanking regions, purify the PCR products and ligate
them to form a deletion construct. Transform the deletion construct into competent P.
aeruginosa PAO1 cells and select for successful recombinants on LB agar plates.

##### b) Preparation of pHERD20T-aiiA /wspF Plasmid:

* Clone aiiA and wspF genes into pHERD20T vector using restriction enzymes and T4 DNA
  ligase.
* Transform the ligated plasmid into competent E. coli cells and select for successful
  transformants on LB agar plates supplemented with carbenicillin.
* Extract the recombinant plasmid from positive E. coli colonies using a plasmid
  extraction kit.

##### c) Electroporation into P. aeruginosa PAO1:

* Prepare competent P. aeruginosa PAO1 cells as described in the prior protocol.
* Mix 1-2 µl of the pHERD20T recombinant plasmid with 50 µl of competent P. aeruginosa
  PAO1 cells.
* Electroporate the mixture and recover the cells in SOC medium, followed by selection
  on LB agar plates supplemented with carbenicillin.

##### d) Verification of Gene Incorporation:

* Perform colony PCR to confirm the integration of aiiA and wspF genes into the P.
  aeruginosa PAO1 genome.

Here, we have proficiently engineered the pf4 mutant strains with the introduction of
high-copy number plasmids pHERD20T-aiiA /wspF (Figure 3).

![4](https://static.igem.wiki/teams/4747/wiki/3-wet-lab/3-3-results/04.png)

#### 3. Integration of aiiA and yhjH Target Genes into the PAO1 Genome through Homologous Recombination

![4](https://static.igem.wiki/teams/4747/wiki/3-wet-lab/3-3-results/05.png)

Initially, we employed PCR to selectively amplify our designated genetic targets, yhjH and
wspF , followed by purification of the amplified products (Figure 5). Custom synthesis of the
genetic constructs for aiiA and ytnP , inclusive of their respective homologous arms, was
performed by Sangon Biotech. Subsequent steps involved the assembly of genetic material
through the Gibson assembly technique and its introduction into DH5α competent cells
utilizing the heat shock method. Verification of successful construct integration was
conducted using PCR and electrophoresis assessments (Figure 6). The culmination of the
process saw the successful conjugation of yhjH and aiiA in the PAO1 genome, confirmed
through further PCR validations (Figure 7).

![4](https://static.igem.wiki/teams/4747/wiki/3-wet-lab/3-3-results/06.png)

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

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Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## 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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