iGEM 2024 — Team im-panama

## **IM-PANAMA Notebook**

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## **Engineering Design Cycle**

Our team often used the design cycle when it came to making changes to our projects. This design process was helpful for our two-year project, since we continued the project from last year we were able to make developments and keep track of them using the design cycle.

![](https://static.igem.wiki/teams/5394/dbtl1-1.webp)

### **Cycle 1 (2023)**

#### **Design**

At the start of 2023, our team began the design and development of a solution to Panama's wilt disease. Our country is a significant banana exporter in Latin America, contributing so much to our economy yet there is a fungus that has corrupted the production and cultivation of plantains. Our project addresses the issue of the Panama wilt disease caused by fusarium oxysporum pathogenic fungus. This fungus clogs the plant xylem vessels leading to water and nutrient deprivation defoliation and that's the plant. the initial design was to create a bioluminescent molecule that would produce a signal once the f. oxysporum was detected.

#### **Build**

Our initial design for our project was the detection of fusarium oxysporum through quorum sensing of Rhizobium leguminosaurum using E. coli as a detection model. Our solution involved genetically modifying bacteria for early detection of the fusarium oxysporum. A genetic circuit with fusaric acid as an inducible promoter was activated by fusaric acid produced by the fungus. We managed to create two genetic circuits that would work together to further develop and detect the signal from the f. Oxysporum.

#### **Test**

During the 2023 cycle of iGEM, we were unable to implement the solution due to the delaying materials and the different scheduling that our team had for the lab work. We were able to produce an efficient circuit yet we were unable to complete the lab work or to implement the circuit we had with actual chemicals. This test period or lack of a test period would become a future learning factor for the team.

#### **Learn**

After all the complexities and developments we've done we got feedback from our project for the last year cycle. Some of our learning from the judging feedback we received last year included that we had to submit our sources and our materials on time to be eligible for more medals. we also needed to put in mind the final consumer product that farmers would end up using, and improving the Practical use for the tech that we're developing. Another Improvement is making smaller prototypes or viable products so we can start testing their functionality. The absence of results from the lab work made it difficult for people to develop good conclusions about our project, this showed us that we need more constructive work we need better protocols, and more developed lab work so we can gather data to present. With all this valuable feedback from last year's session, we were able to start preparing the sign for the 2024 cycle.

### **Cycle 2 (2024)**

#### **Design**

At the beginning of 2024, our team focused on designing a solution for the ongoing threat of Panama wilt disease caused by Fusarium oxysporum. Recognizing that Panama is a vital banana exporter in Latin America, we aimed to develop a bioluminescent detection system that would signal the presence of the pathogen before significant crop damage occurs. Our design incorporates genetically modified E. coli that can produce a bioluminescent signal in response to fusaric acid, a byproduct of the fungal pathogen, utilizing a quorum sensing mechanism to ensure sensitivity and specificity in detection. We also prioritized gathering a strong hardware, math, and finance team to accomplish more in the project.

#### **Build**

In this phase, we genetically modified two bacterial strains to create a dual genetic circuit responsive to fusaric acid. The first strain uses a fusaric acid-inducible promoter to activate quorum-sensing pathways, allowing the bacteria to communicate through acyl-homoserine lactones (AHLs). The second strain is engineered to produce bioluminescence upon activation by AHLs, providing a visual indicator of F. oxysporum's presence in soil samples. The integration of these circuits aims to enhance the overall detection sensitivity by ensuring a strong bioluminescent response as the bacterial population increases.

#### **Test**

Throughout the testing phase, we encountered challenges related to material procurement and scheduling conflicts that hindered our ability to conduct laboratory experiments. Despite successfully developing our genetic circuits, we were unable to implement and test them in a practical setting. This lack of testing has highlighted the importance of timely material acquisition and scheduling, emphasizing the need for a more organized approach in our future work. We also recognized the necessity of establishing clear protocols for successful lab execution. There is enough time to test the hardware’s functionality but not its interaction with the circuit.

#### **Learn**

Feedback from last year's project has been instrumental in shaping our approach for the current cycle. Key insights include the need for timely submission of sources and materials to enhance our eligibility for awards. Additionally, we learned the importance of focusing on the end-user—farmers—and ensuring that our technology is practical and accessible. The importance of developing smaller, functional prototypes for early testing was also emphasized. The absence of conclusive lab results last year underscored the need for more effective protocols and structured data collection methods. Armed with these lessons, we are better equipped to refine our project in the 2023-2024 cycle.

#### **Next Steps**

With the gathered insights, our immediate focus will be on finalizing our genetic constructs and establishing a clear timeline for laboratory testing. We will prioritize the development of user-friendly detection systems that can be easily implemented in agricultural settings, ensuring that our solutions are not only innovative but also practical for the farmers who rely on them. Even after the end of this year’s competition, we feel that it is necessary to wrap up this project and be able to implement this is real life.

## **IM-PANAMA DRN Project Protocol**

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## **Insertion of DNA Into E. Coli Bacteria to Cause Bioluminescence**

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### **Objectives of the Experiments**

Our experiments had the main objective of **inserting DNA into E. coli bacteria;** this bacteria with the inserted DNA was mixed into the soil causing **an emission of bioluminescence in case of the presence of AHL in the soil.** This bioluminescence is what would be detected by our hardware to signal the presence of *Fusarium oxysporum.*

Specifically, we used **genetically engineered plasmid DNA** to be placed inside of the bacteria’s genetic code.

![](https://static.igem.wiki/teams/5394/na005-0100-01-1.webp)

We started by extracting the plasmids with a MiniPrep procedure and creating copies of the plasmid DNA through PCR.

### **E. Coli**

*Escherichia coli (E. coli)* is abundantly found in the soil of subtropical and tropical environments - bananas grow in these kinds of environments.

E. coli is also a model organism, meaning that there are many available studies available for the bacteria. This makes *E. coli* an ideal candidate for our experiments. The strain of *E. coli* that we used, DH5a, was particularly useful to us because of the ample research conducted on it. The strain is proven to have a high transformation efficiency and yield of DNA, this gene is likely to be the most productive.

But how did we introduce DNA into *E. coli*?

![](https://static.igem.wiki/teams/5394/electroport.webp)

This was done through **electroporation** of the *E. coli* cell membrane and the insertion of plasmid DNA into the temporary pore.

We confirmed the presence and stability of the plasmid DNA inside of the bacteria through 3 steps.

1. Agarose gel electrophoresis - we placed amplicons (small pieces of DNA) inside of an electrophoresis device.

The device consists of a gel that has an electric current flowing through it; smaller amplicons move through the gel faster and end up further right while the larger amplicons move through the gel more slowly and end up on the left side of the device. We compared what the supposed location of the amplicons are to where our amplicons were really located on the electrophoresis device to decipher whether the sequence was accurate. We repeated this process with the new plasmid copy to ensure that there are no post-transcriptional mutations in the original sequence. - We performed cell lysis and the Bradford Method - after incubation, we broke down the bacteria’s cell membrane and used polyacrylamide gel electrophoresis to separate the proteins. We identified the plasmid DNA through the Bradford Method.
- We put the *E. coli* in contact with chitin, which is confirmed to induce the expression of bioluminescence in the bacteria.

![](https://static.igem.wiki/teams/5394/screenshot-2023-10-12-at-10-05-31-am.png)

### **Gene Expression Assay**

Main objective: to find the most effective *E. coli* strain as well as the most effective genetic circuit for plasmid DNA insertion.

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 by using the DH5a *E. coli* strain and a genetic circuit, both already employed and validated in synthetic biology assays.

#### **Preparation**

1. Obtain the DH5a bacterial strains.
2. Prepare one genetic circuit with 1 variation and one genetic circuit with 2 variations.
3. Prepare Petri dishes with Luria-Betrani (LB) Broth.

#### **Procedure**

1. Inoculate each strain with a respective genetic circuit in LB medium.
2. Incubate 72 hours under appropriate conditions: in the dark at 21℃.
3. Conduct a test for optical density using a spectrophotometer.
4. Record gene expression levels for each replicate and condition.
5. Conduct test for fluorescence level through electrophoresis.
6. Record gene expression levels for each replicate and condition.

#### **Genetic Circuit Testing**

**Test for Genetic Circuit #1 (tested with 1 variation)**

* The DH5a bacterial strain will undergo 12 tests

**Test for Genetic Circuit #2 (tested with 2 variations)**

1. Variation 1: containing a non-mutated LuxB genetic sequence
2. Variation 2: LuxB sequence combined with Venus Mutation; meant to amplify the bioluminescence of the *E. coli* when in contact with AHL

* The DH5a bacterial strain will undergo 12 tests for each variation of the genetic circuit for a total of 24 tests.

**Negative Controls**

* To understand how the bacteria behaves under natural circumstances, the *E. coli* will grow inside of an LB broth without the genetically engineered plasmid.
* Demonstrating how the medium behaves under natural circumstances without the deliberate growing of bacteria, one sample will be the LB broth with no bacteria on it.

The total number of tests, including those for both genetic circuits and the negative controls will be 38.

**Analysis of Variance**

A statistical analysis software, called ANOVA, will analyze the statistically significant differences among the various treatments.

### **Growth Kinetics Assay**

The main objective of the assay is to stimulate the pathogenic effects of *Fusarium Oxysporum* through the contact of chitin in order to obtain the kinetics rate of the *E. coli.*

The Growth Kinetics Assay will help validate the mathematical model created by our math team.

The 1st circuit and the second circuit were analyzed as a whole unit.

The 2nd genetic circuit was tested with two variations of the LuxB

1. Variation 1: LuxB
2. Variation 2: LuxB combined with Venus Mutation; meant to amplify the bioluminescence of the *E. coli* when in contact with AHL

**Important Notes**

* Experiments will be performed in vitro as the pathogens may pose health risk to lab workers.
* Plants produce antibodies against pathogens like *Fusarium oxysporum.* Chitin is one component of the cell walls in *F. fusarium.* A study tested a synthetic antibody against chitin (as the presence of chitin would mean the presence of the pathogen). The synthetic antibody binds to the chitin, which allows researchers to determine the amount of chitin. The amount of chitin used in the study should be proportional to the amount of fusaric acid produced by *F. fusarium.*

#### **Preparation**

Material for Preparation

* 3 different concentrations of chitin: 10 mg/ml 1 mg/ml 0.1 mg/ml
* Plasmid DNA encapsulated in lipofectamine
* 100 μL of culture with 1x 10^7 of DH5a E. coli strain

**Preparation of Materials for Kinetics Test**

1. Samples (including the chitin and *E. coli* culture) incubated in the dark at 21℃ for 72 hours
2. *E. coli*washed in 1% Tryptone
3. Fluorescence tests conducted on samples

**Preparation of Samples**

1. Test of the 3 concentrations of chitin on DH5a *E. Coli* strain #1 (DH5a)
2. Repeat the test 3 more times for a total of 4 trials.
3. Cell growth and fluorescence measured for each trial, every hour for up to 16 hours after the end of the trial.

**Controls**

* 3 bioreactor tubes with the LB Broth and strain.
* All circuit combinations.
* LB Broth with no bacteria

#### **Procedure**

1. Each strain inoculated in LB medium and LB medium with genetic circuits.
2. Growth monitored by measuring optical density (OD) at regular intervals using a spectrophotometer.
3. OD values recorded for each replicate and condition.
4. Bacteria placed inside of bioreactors with different concentrations
5. Samples placed in a bioreactor to measure OD.
6. Changes in fluorescence observed over time.

Each test has 12 replicates.