iGEM 2024 — Team unt

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{% block title %}Notebook{% endblock %}
{% block lead %} This is our project notebook, read through this page to see how the science happened!{% endblock %}
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## What should this page contain?

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## 5/28/24

##### Elle

- Resuspension of all acquired DNA parts and primers. Made working concentration of primers and amplified two parts (eilRPjexD.5 and PJexD.5-rfpterm) needed to construct our PER1 plasmid containing the EilR repressor and RFP as a reporter gene.
- Also attempted to amplify vector backbone from stored pBMTL-2 cell stock.

## 5/29/24

##### Elle

- Checked results of PCR amplification on gel, eilR and rfp amplification was succesful and bands were acquired, pBMTL-2 vector backbone was not.
- Began an overnight culture in order to miniprep the pBMTL-2 from

## 5/30/24

##### Elle

- Miniprepped the overnight culture of e. coli containing the pBMTL-2 plasmid.
- Ran a gel to determine whether or not the plasmid backbone was succesfully isolated, it was not.

## 5/31/24

##### Elle and Drake

- Used the miniprep for another PCR reaction and increased the extension time, succesfully isolated the backbone via gel confirmation.
- Used Dpn1 restriction digest on the backbone, left over the weekend.

## 6/3/24

##### Elle

- Cleaned the digestion of the pBMTL-2 vector, nanodropped to determine concentration and purity. Results were not satisfactory.
- Reamplified the vector from the stored miniprep, as well as the eilR and RFP inserts. Ran on gel to determine succesful isolation/amplificaiton. Gel confirmed that we had desired products.
- Dpn1 digest of the pBMTL-2 vector PCR product overnight.

## 6/4/24

##### Elle

- Cleaned the Dpn1 digestion, nanodropped for concentration and purity. Came back with 8.7 ng/ul. Also measured the concentration of the inserts.
- Did calculation and set up Gibson Assembly, transformed Gibson into Dh10b competent cells, and then spread on selection plates.
!['Elle and Corbin Gel](https://static.igem.wiki/teams/5273/elle-and-corbin-gel-stuff-and-things.jpeg)

##### Drake

- Performed PCR reactions to amplify pBMTL-2 vector, eilR and RFP inserts.

##### Corbin

- Cleaned backbone and inserts with MONARCH cleanup kit. Nanodropped all three fragments to find concentrations. Performed Gibson Assembly of the three fragments. After Gibson Assembly, transformed D10 cells and plated for overnight growth.

| Components | Volume |
| --- | --- |
| Forward Primer | 1.25uL |

## 6/5/23

##### Elle

- Set up a colony PCR for determination of succcesful plasmid incorporation into competent cells.
- Checked PCR products on gel.- - Gibson Assembly and transformation did not work, as shown in the below image.
    ![First Colony cPCR](https://static.igem.wiki/teams/5273/cpcr-failed.jpeg)

    ## 6/6/23

    ##### Elle

    - Fill in here

    ##### Corbin

    ## 6/7/23

    ##### Elle

    - Fill in here

    ##### Corbin

    ## 6/8/23

    ##### Elle

    - Fill in here

    ## 6/10/23

    ##### Elle

    - Fill in here

    ##### Corbin

    ##### Drake

    - Created Gibson Assembly with pBMTL-2 vector, eiLR and RFP inserts, and conducted transformation protocol to insert plasmid into DH10B competent E. coli cells.

    ## 6/11/23

    ##### Elle

    - Fill in here

    ##### Corbin

    ## 6/12/23

    ##### Elle

    - Fill in here

    ##### Corbin

    ## 6/13/23

    ##### Elle

    - Fill in here

    ##### Corbin

    ## Inspirations

    ---

    * [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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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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#### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

---

Please see the [2024 Medals Page](https://competition.igem.org/judging/medals) for more information.

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{% extends "layout.html" %}
{% 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?

---

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](https://parts.igem.org/Main_Page).

---

Drake

Utilized the protocol below as a schematic for allelic exchange mutagenesis in *Methylococcus capsulatus* strain Bath and inducibility by Cystal Violet dye.

[View PDF](https://drive.google.com/file/d/1MX-UHKrjMg6kiIaadZRe1LpihBpGXacD/view?usp=sharing)

## Inspirations

---

* [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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{% extends "layout.html" %}
{% 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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Due to time constraints and issues with prepared screening methods, we were unable to obtain any data that was not purely preliminary. We were able to finish most of the cloning of our lab-built promoters containing the Jungle Express design elements, but were not able to test the constructs in methanotrophs. For the most part we were able to verify that there was indeed no baseline expression when the Jungle Express system was not induced and that the system can be applied to native promoters and still provide tight transcriptional control. We also managed to verify that the system reaches full induction when induced at micromolar concentrations with Crystal Violet. Those conclude the preliminary results of our project, but due to a lack of significant data there will be no other results published on this page.

## 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 and what you learned from them. 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. Furthermore, you can still impress the judges by thoughtfully discussing what went wrong and how you might change things.

## Inspirations

---

* [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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