iGEM 2023 — Team ispanama

| Date | Progress | Contribution |
| --- | --- | --- |
| January 17, 23 | Talking to more experts specific to synthetic biology, ran surveys/research about Panama's stance on GMO. | STEM : Sohee, Ria, Justina, Carolina, Nicole, Karol, Eitan, Isa, Francisca, Marina, Maria Isabella Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| January 24, 23 | Research about the fungus & agriculture of Panama. | STEM : Sohee, Ria, Justina, Carolina, Nicole, Karol, Eitan, Isa, Francisca, Marina, Maria Isabella Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| January 31, 23 | Brainstorming & Research. | Recycling: Karol, Sohee, Isa Agrobacterium : Carolina, Gabriel, Eitan |
| Febuary 07, 23 | Takeaway from 2022 iGEM winners project & Instagram post about the team. | Plastic : Isa, Carolina, Justina, Ria Chagas : Karol, Maria Isabella, Sohee, Seoyun |
| March 01, 23 | Plan for iGEM bootcamp & go over summary of ideas. | STEM : Sohee, Ria, Justina, Carolina, Nicole, Karol, Eitan, Isa, Francisca, Marina, Maria Isabella Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| June 14, 23 | Registration at iGEM website, finalize research and work on genetic circuits. | STEM : Sohee, Ria, Justina, Carolina, Nicole, Karol, Eitan, Isa, Francisca, Marina, Maria Isabella Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| July 19, 23 | Research about quorum sensing, banana SAR response, work on wiki. | STEM : Sohee, Ria, Justina, Carolina, Nicole, Karol, Eitan, Isa, Francisca, Marina, Maria Isabella Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| July 20, 23 | Data 5Feedback from Mr.Ediner and Professor Gesabel. | STEM : Sohee, Ria, Justina, Carolina, Nicole, Karol, Eitan, Isa, Francisca, Marina, Maria Isabella Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| July 27, 23 | Final deadline for team registration and individual safety forms. | STEM : Sohee, Ria, Justina, Carolina, Nicole, Karol, Eitan, Isa, Francisca, Marina, Maria Isabella Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| August 08, 23 | Abandoning the idea of banana vaccine, only work in detection & quorum sensing part. | STEM : Sohee, Ria, Justina, Carolina, Nicole, Karol, Eitan, Isa, Francisca, Marina, Maria Isabella Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| August 12, 23 | Research about quorum sensing, LuxI and AHL. | STEM : Sohee, Ria, Justina, Carolina, Nicole, Karol, Eitan, Isa, Francisca, Marina, Maria Isabella Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| August 14, 23 | Research about promoters and selected chassis. | T-Shirt updates: Ariel Coding updates: Martina, Gabriel's friend Email to sponsors: Gabriel |
| August 15, 23 | Feedback from Ediner about promoters, genetic circuits. | STEM : Seoyun, Eitan, Isabel, Yeon Woo and Karol Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| August 20, 23 | Discussed the potential chassis, worked on the wiki page, had some coding updates and worked on the sponsors. | STEM : Seoyun, Eitan, Isabel, Yeon Woo, Karol Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| August 22, 23 | Work on genetic circuits, starting on Wiki. | T-Shirt updates: Ariel Coding updates: Martina, Gabriel's friend Email to sponsors: Gabriel |
| August 30, 23 | Research modified versions of LuxR, look for plasmids, fixing the scar part, adding reporter proteins. | STEM ISP : Seoyun, Eitan, Isabel, Yeon Woo, Karol STEM MET : Gaby, Armaan, Isa, Samuel, Valeria |
| September 19, 23 | Worked on the wiki and the project overall, reviewed the final safety form to submit. | STEM : Seoyun, Eitan, Isabel, Yeon Woo, Karol Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| September 20, 23 | Worked on the lab and updated the wiki. | STEM : Seoyun, Eitan, Isabel, Yeon Woo, Karol Non-STEM : Gabriel, Hannah, Yewon, Martina, Ariel, Maia |
| October 4, 23 | Update Meeting for MET members with Ediner. | STEM ISP : Seoyun, Eitan, Isabel, Yeon Woo, Karol STEM MET : Gaby, Armaan, Isa, Samuel, Valeria |
| October 9, 23 | Worked on the Wiki. | WIKI : Seoyun, Ho June, Jordan, Yeon Woo, Eitan, Ariel, Gabriel |


# Gene Expression Assay

The goal of completing a gene expression assay is to determine the best strain and circuit in regards to gene expression. For the validation, standardization, and development of the mathematical model associated with the gene expression system, we aim to identify and standardize the expression conditions using at least two variables. The first variable is based on the evaluation of a minimum of three expression strains that have been previously employed and validated in synthetic biology assays: E. coli BL21, DH5alpha, and K12. Each strain will undergo a minimum of 12 repetitions for each treatment. The second variable under investigation will encompass four types of genetic circuits. Each genetic circuit features a variant of the LuxR protein with characteristics that have been previously identified in the literature or have undergone prior validation of their functions.

Hence, the first circuit incorporates a system validated in E. coli by the iGEM team. The second and third circuits exhibit increasing affinity and stability, respectively. Meanwhile, the fourth circuit integrates characteristics through protein engineering, involving LuxR2 and LuxR3.

Consequently, the three E. coli strains will be subjected to the four circuits with LuxR variants to identify the optimal detection system based on the efficiency and stability of LuxR. The assays will involve cultivating the three studied strains for up to 12 hours, followed by induction with chitin to activate the biosensor. This will enable measurements of optical density (OD) to assess growth, as well as the measurement of fluorescence associated with reporter proteins within each circuit.

## Setup

**1.1. Prepare the bacterial strains:** Obtain four strains - control Lux R, better specificity, better stability, and a combination of specificity with stability.

**1.2. Prepare genetic circuits:** Prepare Genetic Circuit 1 (with one variation) and Genetic Circuit 2 (with four variations).

**1.3. Prepare petri dishes with LB medium.**

## Gene Expression Assay

The 1st Genetic Circuit is tested with one variation for each strain.

**12 replicas x 3 strains = 36 tests**

The 2nd genetic circuits are tested with one positive control and 3 other versions of LuxR:

* V1: A positive control that shows what would typically happen with a previous iGEM genetic part containing Lux R.
* V2: A part designed to focus on transcribing specific parts of the DNA.
* V3: A part designed to be more stable and not overload the circuit system.
* V4: A part combining aspects of the previous 2 parts to ideally produce the most effective Lux R region.

Upon completion, we anticipate conducting 12 replicates for each of the 4 genetic circuits and across 3 strains, summing up to 144 tests. This will enable us to statistically evaluate the data by utilizing an analysis of variance (ANOVA) to determine the presence of any significant differences between the treatments.

There are four negative controls: 3 of which consist of the LB medium and the strain of the bacteria without a genetically engineered plasmid to observe how the bacteria grows without it; 1 consists of just the LB medium in order to have a baseline measurement of wavelength through a spectrophotometer, which provides a base value to compare the turbidity and therefore density of the samples. This comparison will be conducive to concluding which strain-circuit combination grew more. This will result in 12 negative controls.

144 + 36 + 12 = 192 replicates.

The other test we are conducting is fluorescence testing, which will involve electrophoresis in Polyacrylamide Gel.

![Gene Expression Assay Image](https://drive.google.com/uc?export=view&id=13onIHJQjKVKRgDBMkFJ4knCjF_gQBexS)

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## 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.

## 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)

{% endblock %}