iGEM 2025 — Team rum-uprm

{% extends "layoutdeep\_page.html" %}
{% block title %}Notebooks{% endblock %}
{% block header %}

# Notebook

{% endblock %}
{% block page\_content %}

## The Importance of Maintaining Records

---

To maintain laboratory records, we used Benchling. During the summer, the Biology Team worked on becoming familiar with lab techniques and cloning strategies. In late July, we conducted initial tests with RDX, which led us to modify our prototype. In early September, we repeated the RDX tests, confirming that detection worked as expected due to the observed fluorescence. All of our experiments, results, and thought processes are documented on the Results and Engineering Success pages.

Your browser does not support iframes. Please [download the PDF](https://static.igem.wiki/teams/5905/wikipdfs/notebook-benchling-compressed.pdf) to view it.

[*Download Notebook Protocol PDF*](https://static.igem.wiki/teams/5905/wikipdfs/notebook-benchling-compressed.pdf)

{% endblock %}

{% extends "layoutdeep\_page.html" %}
{% block title %}Engineering{% endblock %}
{% block header %}

# Engineering Success

{% endblock %}
{% block page\_content %}

## Engineering Beyond Boundaries: Our RDX Journey

---

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle.webp)

Welcome to our step-by-step journey. Guided by the Design-Build-Test-Learn (DBTL) cycle, we approached each phase systematically to tackle our challenges and develop a reliable, innovative solution.

### How we did it:

[##### Cycle 1:

Functional Consolidation](#Cycle1)

[##### Cycle 2:

Promoter Replacement and Riboswitch Control](#Cycle2)

[##### Cycle 3:

Promoter Optimization](#Cycle3)

[##### Cycle 4:

Construct Synthesis](#Cycle4)

[##### MOVING FORWARD](#Cycle5)

## Functional Consolidation

---

##### Design

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-design.webp)

Our previous RUM-UPRM 2022 team designed three devices: a detection device, a degradation device, and a killswitch device. The detection device included an inducible promoter activated by RDX, the *luxI* gene, which produces the AHL autoinducer molecule, and a reporter gene. The purpose of AHL was to bind to the LuxR protein to form a complex. The AHL-LuxR complex controls the activation of the inducible promoter present in the degradation device, which includes the degradation enzymes *xplA* and *xplB*, along with a reporter gene. The regulation between these two devices was highly refined: degradation would only be triggered after detection of the target molecule (RDX), and AHL served as a means of quorum sensing to coordinate the response across the bacterial population.

While analyzing the complexity of the sequence, we realized that maintaining large plasmids in bacteria is challenging. Bacteria tend not to preserve plasmids that do not provide a survival advantage, especially in the absence of selective pressure, due to the metabolic burden associated with plasmid maintenance [1]. In addition, the size also impacts the transformation efficiency. To address this, we analyzed the effect of plasmid size on transformation efficiency based on the transformation probability reported by [2]. According to their results, transformation efficiency decreases roughly inversely with plasmid size; for example, a 10 kbp plasmid is predicted to have approximately 40% of the efficiency of a 4 kbp plasmid under standard transformation conditions. Based on this, we aimed to reduce the total size of our construct to around 10 kbp to maintain reasonable transformation efficiency. After analyzing the combined sequence length of our three devices, we concluded that the detection and degradation devices could be merged into a single construct, thereby reducing complexity and plasmid burden.

##### Build

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-build.webp)

We removed the detection device while retaining the degradation and kill-switch devices. In this redesign, the degradation device now incorporates the *hmp/hcp* fusion promoter from the former detection device, along with the enzyme-coding genes *xplA* and *xplB* and the reporter from the original degradation device (**Figure 1**). This effectively combines detection and degradation into a single ‘two-in-one’ device. The kill-switch device remains unchanged.

![](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-1-engi.webp)
![](https://static.igem.wiki/teams/5905/images/devices/device-2-killswitch.webp)

**Figure 1.** Modified degradation device and original kill-switch, adapted from the RUM-UPRM 2022 team.

##### Test

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-test.webp)

We assembled both devices in Benchling to determine the total length, which was around 10 kbp. We also analyzed all the parts to ensure that the devices would work without AHL and LuxR. Although AHL, through quorum sensing, would provide coordinated activation at the population level—ensuring that degradation only begins once a sufficient number of cells detect the target molecule—this layer of regulation can be omitted for this stage of the project. In our design, the *hmp/hcp* fusion promoter directly controls the expression of the degradation enzymes (*xplA* and *xplB*) and the reporter gene. This allows each cell to autonomously respond upon detecting the target signal, triggering degradation without requiring intermediate signaling molecules. While this approach sacrifices the population-level synchronization offered by quorum sensing, it simplifies the construct, reduces plasmid size, and enables a faster, direct response, which is suitable for the proof-of-concept stage of our project.

##### Learn

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-learn.webp)

Despite the sophistication and innovative structure of the original devices—particularly the use of layered regulatory control via AHL and LuxR—we realized that this level of complexity was not practical for our current goals. Regulatory precision is important, but the simpler the system, the more robust and reproducible it becomes. By simplifying the design, we increased the feasibility of implementation in real-world settings while retaining the core functionality of detection and degradation.

## Promoter Replacement and Riboswitch Control

---

##### Design

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-design.webp)

After consolidating our device, we realized that the *hmp/hcp* fusion promoter lacked the necessary regulatory regions for proper RDX detection, and our search for other RDX-inducible promoters did not identify any viable alternatives. To ensure stable and predictable expression, we chose the constitutive promoter J23119 from the Anderson family, which has been extensively characterized across different cellular contexts. To provide regulation, we incorporated a riboswitch-based biosensor of RDX, capable of detecting RDX at concentrations as low as 0.44 μmol/L [3]. The concept of incorporating a riboswitch had been suggested by the 2022 RUM-UPRM team, but it was never implemented.

##### Build

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-build.webp)

The degradation device was reconstructed using the J23119 constitutive promoter, ensuring stable expression of all coding sequences (**Figure 2**). The translational riboswitch was placed upstream of each gene to regulate translation in response to RDX (**Figure 3**). The kill-switch device remained unchanged.

![](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-2-engi.webp)

**Figure 2.** Genetic construct with the J23119 constitutive promoter from the Anderson collection.

![](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-3-engi.webp)

**Figure 3.** Illustration of the Genetic Circuit with Riboswitch

##### Test

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-test.webp)

We verified all regulatory regions of the J23119 promoter, including the -10 and -35 boxes, through sequence analysis to confirm functionality.The riboswitch was incorporated into the Benchling-assembled prototype, where both devices were combined into a single plasmid. The final construct had a total sequence length of approximately 11 kb—a size compatible with efficient transformation (by electroporation) and bacterial growth.

##### Learn

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-learn.webp)

Promoter selection and regulatory control are among the most challenging aspects of genetic system design. Using the constitutive J23119 promoter provided stable baseline expression, while the riboswitch allowed RDX-dependent activation. This combination facilitated functional testing and demonstrated how integrating constitutive and inducible elements can improve system predictability and control.

## Promoter Optimization

---

##### Design

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-design.webp)

After deep analysis of the constitutive promoter BBa\_J23119, we realized that it was too strong for our circuit, in terms of transcriptional activity and in exceeding the host’s cellular capacity. By analyzing all the promoters, we observed a linear relationship between promoter strength and cellular burden (**Graph 1**). Based on these considerations, we aimed to identify a promoter that would maintain the metabolic burden within an acceptable range while providing medium to high transcriptional activity. Consequently, we narrowed our selection to two candidates for experimental testing: BBa\_J23118 and BBa\_J23100.

##### Build

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-build.webp)

We used Benchling to assemble each promoter into the circuit and literature data was consulted to guide selection and testing for the most suitable promoter for our prototype.

##### Test

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-test.webp)

Promoter testing included comparison of both transcriptional strength and associated metabolic burden. As shown in **Table 1**, both promoters were compared in terms of strength and metabolic burden. BBa\_J23118 has medium strength (~56% of J23100) and imposes minimal burden, whereas BBa\_J23100 is stronger but causes a significantly higher metabolic burden (20% ± 9.9%).

![](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/grafica-1-engi.webp)

**Graph 1.** Analysis of Anderson family promoters according to Austin UTexas *(iGEM 2019)*, showing the relationship between cellular burden and promoter strength.

**Table 1.** Comparative analysis of promoter strength and associated burden values for BBa\_J23100 and BBa\_J23118.

| Part | Promoter Strength | Burden Value |
| --- | --- | --- |
| BBa\_J23100 | 1.00 | 20.0% ± 9.9% |
| BBa\_J23118 | 0.56 | -0.1% ± 3.8% |

##### Learn

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-learn.webp)

After testing, we determined that BBa\_J23118 was more suitable for our prototype. Since our system employs a translational riboswitch, it does not require a promoter with the high transcriptional activity of BBa\_J23100, and using J23118 minimizes the metabolic burden while still providing sufficient gene expression for functional performance.

## Construct Synthesis

---

##### Design

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-design.webp)

Based on the RDX detection and degradation genetic construct, three genetic circuits were planned for synthesis, varying in riboswitches and ribosomal binding sites (RBS) (**Figure 4**).

![](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-4-engi.webp)

**Figure 4.** (A) Detection and degradation genetic construct. (B) Full RDX detection and degradation sequence containing a constitutive promoter, both degradation protein gene sequences (BBa\_K3857002, BBa\_K3670004), *amilGFP* (BBa\_K592010), the synthetic riboswitch described in [3] before every protein-coding sequence, and a terminator. (C) Riboswitch control sequence containing a constitutive promoter, a synthetic riboswitch, *amilGFP*, and a termination sequence.

##### Build

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-build.webp)

The three circuits were sent for synthesis to Integrated DNA Technologies (IDT). However, only the control sequence (**Figure 5**) was received. The other two circuits were not sent due to stunted colony growth, likely caused by:

* Excessive length of the mRNA transcribed from the constitutive promoter.
* Toxicity resulting from the excess of the XplA and XplB degradation proteins.

##### Test

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-test.webp)

Experimental results showed that the control sequence (**Figure 5**) did not affect colony growth, suggesting that:

* Construct length and constitutive promoter were critical factors.
* An excess of the degradation proteins could be toxic to *E. coli.*

This led to the formulation of two hypotheses:

* The sequences were too long, causing stress for the bacteria, especially with a constitutive promoter.
* The RDX degradation proteins, XplA and XplB, were too toxic for the bacteria.

##### Learn

![](https://static.igem.wiki/teams/5905/images/engineeringpage/igem-eng-cycle-learn.webp)

To address these problems, an inducible promoter was selected. Two alternatives from the iGEM Registry of Standard Biological Parts were chosen:

* σ32 Heat Shock promoter (IIT Madras 2008)
* σ38 Stationary Phase promoter

This change allows controlled expression of long or potentially toxic genes, reducing cellular stress and enabling synthesis of the remaining circuits. Five genetic circuits were designed with multiple cloning sites (MCS) in key points to allow modularity between them.

![](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/2order-3.avif)

**Figure 5:** σ38 Stationary Phase inducible promoter, first MCS includes EcoRI and XbaI restriction sites, RDX specific riboswitch, MCherry protein sequence, second MCS includes SpeI and PstI restriction sites ending with BBa\_B0010 termination sequence.

![](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-6-engi.webp)

**Figure 6:** σ38 Stationary Phase inducible promoter, first MCS includes EcoRI and XbaI restriction sites, a basic ribosomal binding site (RBS), MCherry protein sequence, second MCS includes SpeI and PstI restriction sites ending with BBa\_B0010 termination sequence.

![](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-7-engi.webp)

**Figure 7:** First MCS includes EcoRI, NotI and XbaI restriction sites, followed by the *xplB* and *xplA* genes, which encode RDX degradation enzymes, each with the RDX specific riboswitch. The second MCS includes SpeI, NotI and PstI restriction sites.

![](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-8-engi.webp)

**Figure 8:** First MCS includes EcoRI, NotI and XbaI restriction sites, followed by the *xplB* and *xplA* genes, which encode RDX degradation enzymes, each with a ribosomal binding site. The second MCS includes SpeI, NotI and PstI restriction sites.

![](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-9-engi.webp)

**Figure 9:** σ32 Heat Shock inducible promoter, MCS includes EcoRI, NotI, XbaI, SpeI, NotI and PstI restriction sites (standard iGEM prefix and suffix). The circuit is closed with BBa\_B0010 termination sequence.

Two controls were made, one with the riboswitch (**Figure 5**) to simply test for detection. The second control was ordered in case the riboswitch control does not give out a signal. If this second control (**Figure 6**) does not give out a signal either, we can conclude that the mistake was in the *in-silico* assembly of the sequence. However, if the first control does work, we can proceed with the degradation tests by cutting and cloning the sequence in **Figure 7** into the control sequence shown in **Figure 5**. This is why multiple cloning sites were added, so fragments could be added or removed at will. As for the circuit in **Figure 8**, this one was ordered in case the riboswitch gave problems and degradation wanted to be tested without having to detect first. Finally, the sequence shown in **Figure 9** will be used once we upscale our operations to a batch bioreactor for the degradation tests that will be handled by the engineering team.

## MOVING FORWARD

---

![](https://static.igem.wiki/teams/5905/lab-pictures/lab-8.webp)

The only construct that could be successfully synthesized was the one containing the degradation proteins with a ribosomal binding site (**Figure 2**), as the bacterial colonies did not grow well otherwise. However, we tested RDX detection—previously tested in LB with negative results—using the RDX riboswitch in M9 1× media and observed activation. This confirms that Device 1 effectively detects RDX, as indicated by the AmilGFP signal, which turned yellow upon RDX detection. Future experiments are needed to determine the RDX degradation rate and quantify its efficiency. Additionally, studies should investigate the effects of RDX on bacterial growth, since bacteria grown in glucose-free medium with RDX appeared to utilize it as a carbon source. Evaluating both short- and long-term effects will be essential.

##### References

[1] D. K. Summers, "The kinetics of plasmid loss," *Trends in Biotechnology*, vol. 9, no. 1, pp. 273–278, 1991. [Online]. Available: [https://doi.org/10.1016/0167-7799(91)90089-Z](https://doi.org/10.1016/0167-7799%2891%2990089-Z)

[2] D. Hanahan, "Studies on transformation of *Escherichia coli* with plasmids," *Journal of Molecular Biology*, vol. 166, no. 4, pp. 557–580, 1983. [Online]. Available: [https://doi.org/10.1016/S0022-2836(83)80284-8](https://doi.org/10.1016/S0022-2836%2883%2980284-8)

[3] M. L. Mayo, J. O. Eberly, F. H. Crocker, and K. J. Indest, "Modeling a synthetic aptamer-based riboswitch biosensor sensitive to low hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) concentrations," *PLoS One*, vol. 15, no. 11, e0241664, 2020. [Online]. Available: <https://doi.org/10.1371/journal.pone.0241664>

{% endblock %}

{% extends "layoutdeep\_page.html" %}
{% block title %}Experiments{% endblock %}
{% block header %}

# Experiments

{% endblock %}
{% block page\_content %}
Project Description

## **Introduction**

---

This section outlines the experimental workflow used to test and validate the genetic constructs designed for the RDX biosensor project. The experiments aimed to confirm the integrity of the synthesized sequences, establish successful transformation in *Escherichia coli*, and evaluate the bacterial response to the explosive compound RDX. All steps were carried out under standardized molecular biology and biosafety protocols to ensure accuracy, reproducibility, and safety.
The experimental process began when the synthesized DNA sequences were received, containing the riboswitch and reporter elements. These sequences were transformed into *E. coli* DH5α electrocompetent cells using the electroporation method to generate recombinant bacterial strains carrying the target constructs. Transformed colonies were selected on kanamycin-containing agar plates and later verified for correct base pair length through restriction enzyme digestion and agarose gel electrophoresis. These analytical steps confirmed the expected insert sizes and validated the success of the DNA synthesis and transformation processes.
Following verification, the engineered *E. coli* strains were cultured in M9 minimal medium supplemented with kanamycin and exposed to different concentrations of RDX. The goal was to assess whether the riboswitch could detect and respond to the presence of RDX, generating a measurable reporter signal that reflects biosensor activity.
Additionally, protocols for gel extraction and ligation were included as contingency procedures in case the synthesized constructs needed to be subcloned into an alternative vector, such as an iGEM-compatible plasmid. This ensured flexibility for future molecular modifications or standardization with iGEM assembly requirements.
Overall, the workflow combines transformation, molecular verification, and biosensor functional testing to evaluate the potential of the riboswitch-based system for RDX detection. The following sections detail the materials, methods, and adapted Standard Operating Procedures (SOPs) applied throughout the experimental process.

###### Workflow

![workflow](https://static.igem.wiki/teams/5905/experiment-page/workflow.webp)

###### Flowchart

![Flowchart](https://static.igem.wiki/teams/5905/experiment-page/flowchart.webp)

## **Protocols**

---

\* It is important to note that some of the Standard Operating Procedures shown were adapted from already existing ones prepared by undergraduate students working in the **Microbial Biotechnology and Bioprospecting Lab (MBBL)**.

1. Transformation (Electroporation method)

#### Overview

This protocol describes the transformation of plasmids into *E. coli* DH5α electrocompetent cells using electroporation. The transformed cells are then recovered and plated on selective media for subsequent cryopreservation and downstream applications.

![Electrical Transformation](https://static.igem.wiki/teams/5905/experiment-page/protocol-diagram-1.webp)

#### Materials

* Electrocompetent cells: *E. coli* DH5α
* Plasmid DNA: iGEM parts (or other plasmids of interest)
* Electroporator
* Electroporation cuvettes
* SOC medium (pre-warmed to room temperature)
* Nutrient agar plates with appropriate selection antibiotic (Kanamycin for this experiment)
* Pipettes and sterile tips: P1000, P200, P20, and P10
* Sterile 1.5 mL microcentrifuge tubes
* Sterile PCR tubes
* Sterile L-shaped spreaders (hockey sticks)
* Ice bucket with ice
* Incubator shaker (37°C)
* 95% ethanol
* Sterile distilled water

#### Protocol

###### Preparation

*Keep electrocompetent cells, DNA, cuvettes, and SOC on ice at all times.*

1. Clean the electroporation cuvette before use: rinse once with 95% ethanol, then rinse twice with sterile distilled water. Dry the outside thoroughly.

###### Mixing cells and DNA

2. Thaw 20 µL of electrocompetent *E. coli* DH5α cells on ice in a sterile PCR tube.
3. Add 1–5 µL of plasmid DNA (10–100 ng recommended). Mix gently by pipetting. Avoid introducing bubbles.

###### Electroporation

4. Transfer the cell-DNA mixture to a pre-chilled electroporation cuvette. Tap gently to ensure the liquid is at the bottom.
5. Place the cuvette into the electroporator.
6. Set the electroporator to 1.8 kV (1800 V) (for a 1 mm cuvette; adjust if using 2 mm). Use one pulse only.

*Note: A loud pop or spark indicates arcing, often due to excess salts in the DNA. Discard and repeat with cleaner DNA.*

###### Recovery

7. Immediately add 280 µL of SOC medium to the cuvette. Resuspend cells gently.
8. Transfer the suspension into a sterile 1.5 mL microcentrifuge tube.
9. Incubate at 37°C, 200 rpm for 45–60 minutes to allow expression of antibiotic resistance genes.

###### Plating

10. Plate 50–100 µL of the recovered culture onto a selective agar plate (LB agar + kanamycin). Store the rest in 4°C in case no colonies are observed after overnight incubation.
11. Spread evenly using a sterile spreader.
12. Incubate plates at 37°C overnight.

2. Plasmid Digestion (Restriction Digest)

#### Overview

Restriction digestion is used to cut plasmid DNA at specific recognition sites using restriction enzymes, preparing DNA for cloning, ligation, or analytical applications.

![Digestion](https://static.igem.wiki/teams/5905/experiment-page/diagram-protocol-for-digestion-1.webp)

#### Materials

* Plasmid DNA
* PCR tubes (0.2 mL or 0.5 mL)
* Micropipettes and sterile tips
* Ice bucket

#### Reagents & Solutions

* 10X NEBuffer (rCutSmart® Buffer, NEB)
* Restriction enzymes (EcoRI, XbaI, NotI, SpeI, and PstI are some of the enzymes we use in the lab)
* Nuclease-free water

#### Protocol

###### Thaw reagents

1. Thaw buffer and water at room temperature. Keep restriction enzymes on ice at all times.

###### Reaction setup

Typical reaction volume: 10 µL (can be scaled).

| Component | Volume (example 10 µL reaction) |
| --- | --- |
| Plasmid DNA | 100–500 ng (≈1–7 µL, depending on concentration) |
| 10X rCutSmart Buffer | 1 µL |
| Restriction enzyme(s) | 0.5–1 µL each (typically 1–10 units per reaction) |
| Nuclease-free water | To final volume (adjust so total = 10 µL) |

Add components in the following order (to reduce enzyme inactivation):

2. Add nuclease-free water first.
3. Add 10X rCutSmart Buffer.
4. Add plasmid DNA.
5. Add restriction enzyme(s) last.

###### Mixing

6. Gently flick the tube or pipette up and down. Do not vortex.

###### Incubation

7. Incubate at 37°C for 1–2 hours.
8. For high-fidelity (HF) enzymes, 30–60 min is usually sufficient.

###### Optional

9. Heat-inactivate enzymes if recommended (65°C for 20 min, depending on enzyme).
10. Proceed to downstream applications (ligation, gel electrophoresis, etc.).

3. Miniprep

#### Overview

This protocol describes the isolation of plasmid DNA from *E. coli* overnight cultures using the alkaline lysis method, followed by precipitation and purification. The resulting DNA can be used for cloning, digestion, or storage.

![Miniprep](https://static.igem.wiki/teams/5905/experiment-page/mini-prep-diagram-protocol-1.webp)

#### Materials

* Micropipettes (P1000, P200, P10) with sterile tips
* Sterile 1.5 mL microcentrifuge tubes
* Bacterial culture (overnight, grown with appropriate antibiotic)
* Centrifuge capable of 13,000 rpm

#### Reagents & Solutions

* **Solution I (buffer):**
  - 50 mM glucose
  - 25 mM Tris-Cl, pH 8.0
  - 10 mM EDTA
  ***Add 4 mg/mL lysozyme before use.***
* **Solution II (Lysis):**
  - 0.2 N NaOH
  - 1% (w/v) SDS
  ***Prepare this solution immediately before use.***
* **Solution III (DNA precipitation):**
  - 29.5 mL glacial acetic acid
  - KOH pellets added until pH 4.8
  - Add water up to 100 mL
  - Store at room temperature, do not sterilize.
* Chloroform
* Isopropanol 100%
* Ethanol 80%
* 1X TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0)

#### Protocol

###### Culture preparation

1. Inoculate the desired colony into 3–6 mL of nutrient broth with the appropriate antibiotic.
2. Incubate overnight (~16–24 h) at 37°C with shaking.

###### Cell collection

3. Transfer 1.5 mL of culture into a microcentrifuge tube.
4. Centrifuge at 13,000 rpm for 2 minutes to pellet cells.
5. Discard the supernatant.
6. Repeat with another 1.5 mL of culture into the same tube to increase yield.

###### Resuspension

7. Resuspend the pellet in 100 µL of Solution I (cold, with lysozyme).
8. Incubate at room temperature for 5 minutes to allow cell wall digestion.

###### Lysis

9. Add 200 µL of freshly prepared Solution II (cold).
10. Mix gently by inverting (do not vortex).
11. Incubate on ice for 5 minutes.
12. *Note: Solution should become viscous/clear—indicates lysis.*

###### Neutralization

13. Add 150 µL of Solution III.
14. Mix immediately by gentle inversion or brief vortex.
15. Incubate on ice for 5 minutes.
16. Centrifuge at 13,000 rpm for 5 minutes.
17. Carefully transfer the clear supernatant to a new tube.

###### Chloroform extraction

18. Add 200 µL of chloroform to the supernatant.
19. Vortex thoroughly, centrifuge 1 min at 13,000 rpm.
20. Carefully collect the aqueous (upper) phase into a fresh tube.

###### DNA precipitation

21. Add an equal volume of 100% isopropanol.
22. Mix by inversion and incubate at room temperature for 5 minutes.
23. Centrifuge at 13,000 rpm for 5 minutes.
24. Discard supernatant carefully.

###### DNA washing

25. Wash pellet with 200 µL of cold 80% ethanol.
26. Centrifuge 1 min at 13,000 rpm.
27. Discard supernatant completely.

###### Drying and resuspension

28. Air dry pellet no longer than 5 minutes (avoid overdrying).
29. Optional: quick spin to collect residual liquid.
30. Resuspend pellet in 50 µL of 1X TE buffer (or nuclease-free water).

###### Storage

31. Store plasmid DNA at –20 °C.

4. Gel DNA Purification

#### Overview

This protocol describes purification of DNA fragments from low-melting-point agarose gels using β-Agarase I (New England Biolabs). The method ensures recovery of intact DNA for downstream applications such as cloning, ligation, or sequencing.

![Gel DNA Purification](https://static.igem.wiki/teams/5905/experiment-page/gel-dna-purification-diagram-protocol.webp)

#### Materials

* Water bath (set to 65°C)
* Heat block (set to 42°C)
* Microcentrifuge
* Sterile centrifuge tubes (1.5 mL)
* Micropipettes (P1000, P200, P10, P2) with sterile tips
* Ice bucket with ice
* Clean scalpel or razor blade

#### Reagents

* Low melting point agarose
* 1X β-Agarase I Reaction Buffer (NEB)
* β-Agarase I enzyme (NEB)
* NaCl (0.5 M) or alternative salts
* 100% isopropanol
* 70% ethanol (cold)
* TE buffer or nuclease-free water

#### Protocol

###### Excise the DNA band

1. Use a clean scalpel or razor blade to cut the DNA-containing band from the gel.
2. Minimize excess agarose around the DNA band.

###### Equilibration (standard method)

3. Wash the solid gel slice twice with 2 volumes of 1X β-Agarase I buffer on ice, 30 minutes each wash.
4. Discard the buffer after each wash.

###### Melting

5. Incubate the gel slice at 65°C in water bath for 10 minutes until fully molten.

###### Enzyme digestion

6. Cool the molten agarose to 42°C in heat block.
7. Add 1 unit of β-Agarase I per 200 µL of 1% agarose.
8. Incubate at 42°C for 1 hour.

###### Salt adjustment

9. Add salt to achieve one of the following concentrations for precipitation: 0.5 M NaCl, 0.3 M NaOAc, 2.5 M NH4OAc, or 0.8 M LiCl.

###### Pre-clear sample

10. Chill on ice for 15 minutes.
11. Centrifuge at 14,000 RPM for 17 minutes to pellet undigested carbohydrates.
12. Carefully transfer the DNA-containing supernatant to a new tube.

###### DNA precipitation

13. Add 0.7 volumes of 100% isopropanol.
14. Mix thoroughly and chill on ice.
15. Centrifuge at 14,000 RPM for 17 minutes.

###### Washing

16. Remove supernatant carefully.
17. Wash pellet with cold 70% ethanol.
18. Centrifuge 1–2 minutes and discard supernatant.

###### Drying

19. Air dry the pellet at room temperature (avoid overdrying).

###### Resuspension

20. Resuspend DNA pellet in TE buffer or nuclease-free water.
21. Store at –20°C.

5. Gel Electrophoresis

#### Overview

Agarose gel electrophoresis is used to verify DNA size, analyze genetic material, and check the quality/purity of DNA samples. DNA fragments migrate through agarose in an electric field, and are visualized with a nucleic acid stain. Gel electrophoresis is also an important step for DNA insert separation.

![Gel Electrophoresis](https://static.igem.wiki/teams/5905/experiment-page/gel-electrophoresis-diagram.webp)

#### Materials

* DNA sample
* NEB 1kb ladder
* Electrophoresis chamber
* NEB 6x Loading dye
* Ethidium bromide (EtBr)
* TE buffer
* 1X TAE running buffer and for gel preparation
* Agarose
* Microwave
* Power supply
* UV transilluminator
* PCR tubes or parafilm

#### Protocol

###### Prepare agarose gel

1. Weigh 0.3 g agarose and dissolve in 30 mL of 1X TAE buffer.
2. Heat in a microwave until fully dissolved (avoid boiling over).
3. Cool to 60°C, then pour gel into casting tray with comb in place. Allow to solidify (~20–30 min).

###### Set up electrophoresis chamber

4. Place gel into chamber and cover completely with 1X TAE running buffer.
5. Carefully remove comb.

###### Prepare samples

6. Mix DNA with loading dye (this can be done in PCR tubes or on top of a clean piece of parafilm).
7. Load wells with samples: NEB 1kb ladder in one lane and DNA samples in the rest (volume depends on concentration, usually 5–10 µL).

###### Run gel

8. Attach electrodes (DNA runs toward the red/positive electrode).
9. Run at 90 V for 65 minutes.

###### Visualization

10. Soak gel in EtBr solution (0.5 µg/mL) for 15 min, then rinse in water for 10–15 min.
11. Visualize under UV or blue-light transilluminator.

*Safety note:* Ethidium bromide is mutagenic — handle with PPE and dispose properly or use a safer stain alternative.

6. Ligation

#### Overview

DNA inserts with compatible sticky ends are ligated into the plasmid vector using T4 DNA Ligase, following the Ligation Protocol with T4 DNA Ligase by New England Biolabs.

![Ligation Diagram](https://static.igem.wiki/teams/5905/experiment-page/ligation-diagram.webp)

#### Materials

* T4 DNA Ligase
* T4 DNA Ligase Buffer (10X)
* Vector DNA
* Insert DNA
* Nuclease-free water
* Microcentrifuge tubes
* Ice bucket

#### Protocol

###### Reaction setup

1. Prepare the ligation mixture on ice as follows (example: 20 µL total reaction, using a 1:3 vector-to-insert molar ratio):

* X µL Vector DNA
* X µL Insert DNA
* 2 µL 10X T4 DNA Ligase Buffer *(thaw and resuspend at room temperature before use)*
* 1 µL T4 DNA Ligase (added last)
* Add nuclease-free water to final volume

*Note: NEB’s ligation calculator or standard molar ratio guidelines can be used to adjust volumes based on DNA sizes and concentrations.*

###### Mix

2. Gently pipette up and down to mix.
3. Briefly centrifuge to collect contents at the bottom of the tube.

###### Incubation

4. Incubate at 16 °C overnight for maximum efficiency.

###### Heat inactivation

5. Inactivate the enzyme at 65 °C for 10 minutes.

###### Transformation

6. Chill the reaction on ice.
7. Transform 1–5 µL of the ligation mixture into 20 µL of competent cells.

7. Plasmid DNA Purification (ZymoPURE™ Miniprep, Spin-Column Method)

#### Overview

This protocol describes the purification of plasmid DNA from *E. coli* using the ZymoPURE™ Plasmid Miniprep Kit with the centrifugation method. The kit allows rapid recovery of up to 100 µg of high-quality plasmid DNA suitable for downstream applications including cloning, sequencing, and transfection.

#### Materials

* Bacterial culture: 0.5–5 mL overnight *E. coli* culture (LB + antibiotic)
* ZymoPURE™ Plasmid Miniprep Kit reagents:

+ P1 (Red)
+ P2 (Blue)
+ P3 (Yellow)
+ Binding Buffer
+ Wash 1
+ Wash 2 (with ethanol added, if required)
+ Elution Buffer

* Zymo-Spin™ II-PX Columns with collection tubes
* 1.5 mL sterile microcentrifuge tubes
* Microcentrifuge (≥ 10,000 RPM)
* P1000, P200, and P10 micropipettes and sterile tips

#### Protocol

###### Preparation

1. Add the required ethanol to ZymoPURE™ Wash 2 concentrate before use.
2. Equilibrate all reagents to room temperature.
3. Ensure P2 and Binding Buffer are fully dissolved (warm to 30–37°C if precipitated).

###### Cell Lysis & Neutralization

4. Pellet 0.5–5 mL of overnight bacterial culture at full speed (15–20 sec). Discard supernatant.
5. Resuspend pellet in 250 µL ZymoPURE™ P1 (Red) by pipetting or vortexing.
6. Add 250 µL ZymoPURE™ P2 (Blue). Gently invert 8–10 times (do not vortex). Incubate 3 min.
7. *Solution should appear clear, purple, and viscous.*
8. Add 250 µL ZymoPURE™ P3 (Yellow). Invert until solution turns yellow and precipitate forms.
9. Centrifuge 6.5 min at 14,000 RPM.

###### DNA Binding

10. Transfer 600 µL supernatant to a new 1.5 mL tube.
11. Add 260 µL Binding Buffer, vortex 15 sec.

###### Column Purification (Centrifugation)

12. Transfer entire lysate–binding buffer mix to a Zymo-Spin™ II-PX Column in a collection tube.
13. Incubate 1 min, then centrifuge ≥ 10,000 RPM for 1 min. Discard flow-through.
14. Add 800 µL Wash 1. Centrifuge ≥ 10,000 RPM for 1 min. Discard flow-through.
15. Add 800 µL Wash 2. Centrifuge ≥ 10,000 RPM for 1 min. Discard flow-through.
16. Add 200 µL Wash 2. Centrifuge ≥ 10,000 RPM for 1 min. Discard flow-through.
17. Centrifuge column again ≥ 10,000 RPM for 1 min to remove residual wash buffer.

###### Elution

18. Place column into a clean 1.5 mL tube.
19. Add 25 µL Elution Buffer directly to the column matrix. Incubate 2 min.
20. Centrifuge ≥ 10,000 × g for 1 min to elute plasmid DNA.
21. Store DNA at ≤ –20°C.

\* **Note**: the next two protocols were done in the Chemistry Department under supervision of licensed personnel in the Center for Chemical Sensors Chemical Imaging and Surface Analysis Center. One of our team members previously did undergraduate research in this lab and she carried out the following protocols.

8. Preparation of 5.00 mM stock of RDX (MW=222.12 g/mol) in acetone

#### Overview

This protocol describes the preparation of a sterile, accurately quantified 5.00 mM stock solution of RDX (MW = 222.12 g/mol) in acetone at a final volume of 25.00 mL, suitable for experimental use, following institutional safety procedures.

#### Calculations

Desired concentration × volume = moles required

\[ 5.00 \, \text{mM} = 0.00500 \, \text{mol/L} \]

\[ \text{Volume} = 25.00 \, \text{mL} = 0.02500 \, \text{L} \]

Moles = concentration × volume

\[ \text{moles} = 0.00500 \, \text{mol/L} \times 0.02500 \, \text{L} \]

\[ \text{moles} = 0.000125000 \, \text{mol} \]

Convert moles to mass using MW of RDX (222.12 g/mol)

\[ \text{mass (g)} = \text{moles} \times \text{MW} \]

\[ \text{mass} = 0.000125000 \, \text{mol} \times 222.12 \, \text{g/mol} \]

\[ 222.12 \, \text{g/mol} \times 0.000125000 \, \text{mol} = 0.0277650 \, \text{g} \]

Conversion

\[ 0.0277650 \, \text{g} \times 1000 \, \text{mg/g} = 27.7650 \, \text{mg} \]

#### Materials & Equipment

* Analytical balance (readability ≤0.1 mg)
* Amber glass vial or amber glass bottle with tight cap (volume ≥ 50 mL) — acetone compatible
* Volumetric flask and pipettes to make up to 25.00 mL
* Acetone (reagent grade)
* Weighing paper or small weighing boat
* Forceps/tweezers (optional)
* Labels and marker (chemical name, concentration, solvent, date, preparer, hazard info)
* Fume hood

#### Protocol

###### Pre-label container

1. Label volumetric flask: “RDX — 5.00 mM in acetone; 25.00 mL; prepared [date]; prepared by [name]; hazard: [type of hazard]”

###### Weigh compound

2. Tare weighing boat on the analytical balance. Add RDX until the balance reads 27.77 mg. Use tweezers/clean spatula; avoid touching with bare hands. Immediately transfer the weighed solid into the pre-labeled volumetric flask or vial in the fume hood.

###### Add solvent to dissolve

3. In the hood, add approximately 15–20 mL of acetone to the flask/vial to dissolve the solid. (If using a 25.00 mL volumetric flask: add solvent to dissolve then bring to the mark — see next step.) Swirl gently or invert to mix; do not vortex if using open glassware. Do not heat the acetone (it is flammable). If dissolution is slow, gentle swirling is sufficient.

###### Bring to final volume

4. After the compound is completely dissolved, add acetone to bring the solution to the 25.00 mL mark (final volume). If using a graduated cylinder, transfer quantitatively into a 25.00 mL volumetric flask and then adjust to the mark with acetone. Ensure meniscus at calibration line at eye level.

###### Mix & transfer

5. Cap the flask/vial and invert several times to ensure homogeneity. If using a volumetric flask, invert ~10–15 times.

###### Storage

6. Store at 4°C at the recommended temperature. Include hazard label on storage container.

###### Waste & decontamination

7. Dispose of all acetone rinses, contaminated wipes, and weighing boats in the designated organic solvent waste container, following institutional hazardous waste procedures. Do not pour acetone or compound into sink. Decontaminate balance area and hood per lab SOP.

9. Detection of RDX by *E. coli* in M9 medium with kanamycin

#### Overview

Assess whether *E. coli* detects RDX when grown in defined medium with antibiotic selection by comparing biological responses across multiple exposure levels and appropriate controls.

#### Materials

* Sterile culture tubes (suitable volume for 6 mL working volume) and caps
* Sterile pipette tips (sized for planned liquid handling)
* Sterile serological/transfer pipettes
* Sterile inoculating tools
* Chemical fume hood (for handling RDX)
* Incubator and shaker
* Micropipettes (P10, P20, P200, P1000)
* Sterile test tubes

#### Reagents

* M9 defined medium (prepared, sterile)
* Kanamycin stock (1000× concentrated) — label with concentration, lot, date, preparer
* Authorized *E. coli* strain culture (per your lab’s IBC approval)
* RDX stock (amber vial, 5.00 mM in acetone, 25 mL) — properly labeled and with SDS on hand
* Acetone (reagent grade) — if needed for vehicle controls or rinses

#### Protocol

###### Preparation of test tubes

1. Pour 6 mL of previously prepared and sterilized M9 defined medium into desired amount of test tubes.
2. Make sure appropriate aseptic techniques are being employed (work close to Bunsen burner).
3. Add 6 µL of 1000× concentrated kanamycin antibiotic.

###### Inoculation

4. With a sterile inoculation loop, transfer pre-incubated bacteria into test tubes to ensure the same transfer amount.

###### Adding RDX

5. \****Note**: This step was carried out in the chemistry department under supervision.*
6. Transfer calculated volumes of RDX 5 mM stock into test tubes.

| Target [RDX] (µM) | For 6 mL LB Tubes: Volume from 5 mM Stock (µL) | For 6 mL LB Tubes: Volume from 10 mM Stock (µL) |
| --- | --- | --- |
| 0.00 | 0 | 0 |
| 0.10 | 0.1 | 24 |
| 0.30 | 0.4 | 72 |
| 0.44 | 0.50 | 105.2 |
| 0.50 | 0.60 | 120 |
| 1.00 | 1.20 | 240 |
| 2.00 | 2.40 | 480 |
| 3.80 | 4.60 | 912 |
| 4.40 | 5.30 | 1056 |
| 5.00 | 6.00 | 1200 |
| 6.20 | 7.40 | 1488 |

###### Incubation

6. Incubate at 37°C at 160 RPM for 48 hours. Make sure to take note of any observations seen at 24 and 48 hours.

###### Disposal

7. Dispose of medium with RDX in the waste container and follow the laboratories disposal protocols.

## **Appendix**

---

The SOPs listed below were used as the basis for adapting our laboratory protocols and originate from the **Microbial Biotechnology and Bioprospecting Lab (MBBL), led by Carlos Ríos-Velázquez**.

* [*Manual de Genética de Bacterias [Spanish]*](https://static.igem.wiki/teams/5905/wikipdfs/appendixexperiments/manual-de-gene-tica-de-bacterias-revisado-2020.pdf)
* [*RDX–Riboswitch Test Protocol [Spanish]*](https://static.igem.wiki/teams/5905/wikipdfs/appendixexperiments/rdx-riboswitch-test-protocol-crv-review.pdf)
* [*Transformation by Electroporation [Spanish]*](https://static.igem.wiki/teams/5905/wikipdfs/appendixexperiments/sop-s1-2024-2025-coralie-transfor-by-electroporation.pdf)
* [*Cálculos para Reacción con Enzimas de Restricción [Spanish]*](https://static.igem.wiki/teams/5905/wikipdfs/appendixexperiments/sop-s1-2024-2025-kevin-ca-lculos-para-reaccio-n-con-enzimas-de-restriccio-n.pdf)

## **References**

---

[1] C. Hernández Guerrero, “Transformation of *Escherichia coli* electrocompetent cells by electroporation (SOP S1 2024–2025),” unpublished SOP, University of Puerto Rico at Mayagüez, Biology Department, Microbial Biotechnology and Bioprospecting Laboratory, Dec. 2024.

[2] New England Biolabs, “DNA ligation with T4 DNA ligase (Protocol M0202),” [Online]. Available: https://www.neb.com/en/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202. [Accessed: Oct. 4, 2025].

[3] Universidad de Puerto Rico, Recinto Universitario de Mayagüez, Departamento de Biología, “Manual de genética de bacterias,” Rev. 2020, Lab. 3: Extracción de plásmidos, pp. 21–27, 2020.

[4] New England Biolabs, “DNA purification from agarose gels using β-Agarase I (Protocol M0392),” [Online]. Available: https://www.neb.com/en/protocols/0001/01/01/dna-purification-from-agarose-gels-using-beta-agarase-i-m0392. [Accessed: Oct. 4, 2025].

[5] Zymo Research, “ZymoPURE™ Plasmid Miniprep Kit (Instruction manual, Ver. 2.0.5),” Zymo Research Corp., Cat. Nos. D4208T, D4209, D4210, D4211, D4212, 2023.

[6] iGEM-RUM, “RDX–Riboswitch test protocol,” unpublished manuscript, University of Puerto Rico at Mayagüez, Department of Biology, 2025.

{% endblock %}

{% extends "layoutdeep\_page.html" %}
{% block title %}Results{% endblock %}
{% block header %}

# Results

{% endblock %}
{% block page\_content %}
Project Description

Press the button below to view more information.

## Introduction to Results

### **Result Preamble**

---

This year’s the **iGEM RUM-UPRM R-DetoX team** focused on improving the previous project design by reducing the total number of genetic circuits from three — (1) detection, (2) degradation, and (3) kill switch — to two: (1) **detection and degradation**, and (2) **kill switch**. This simplification aimed to increase modularity, reduce metabolic burden on the host cells, and improve overall construct stability.

![rzeka insta](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-3-engi.webp)

**Figure 1**: Detection and degradation genetic construct

Shown above is the proposed improved genetic construct, represented using **SBOL notation**. The sequence includes a **constitutive promoter**, the ***xplA*** and xplB genes responsible for RDX degradation, a **reporter gene (*amilGFP*)**, and a **terminator sequence**. Additionally, a **synthetic riboswitch with an aptamer-based sensor specific to RDX** [1] was inserted before each coding sequence to enable ligand-dependent regulation. Based on this design, **three genetic circuits** were submitted for synthesis, as shown below.

![rzeka lupa](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-2-results-page.webp)

**Figure 2**: Riboswitch control sequence containing a constitutive promoter, the synthetic riboswitch [1], *amilGFP*, and a terminator sequence.

![rzeka lupa](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-3-engi.webp)

**Figure 3**: Full RDX detection and degradation sequence containing a constitutive promoter, both degradation protein genes (**XplA** and **XplB**), ***amilGFP***, and the RDX-specific synthetic riboswitch [3] before each coding sequence, followed by a terminator.

![rzeka lupa](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-4-results-page.webp)

**Figure 4**: Construct identical to that shown in **Figure 3**, except that each riboswitch was replaced with a standard **ribosome binding site (RBS)**.

The three genetic circuits shown in **Figures 2–4** were submitted for DNA synthesis to **Integrated DNA Technologies (IDT)**. Of these, **only one sequence was successfully synthesized and delivered**— the control construct shown in **Figure 2**. This construct was later transformed and cryopreserved for subsequent testing (see Results section).

The other two constructs were not delivered, as the colonies displayed **stunted growth during synthesis**. The main differences between the successful construct and the ones that failed were their increased sequence length and inclusion of **XplA** and **XplB degradation proteins**. We hypothesized that the amount of proteins may have been **toxic to the host bacteria** or that the **longer DNA fragments** imposed excessive genetic expression and physiological metabolic stress. Furthermore, the constructs employed a **constitutive promoter**, which continuously drives transcription; this may have resulted in overexpression of lengthy mRNA transcripts and additional cellular burden, further contributing to the observed synthesis issues.

To address these issues, two hypotheses were proposed: (1) sequence length and continuous transcription imposed excessive metabolic stress, and (2) the degradation proteins were cytotoxic when constitutively expressed. Both challenges could be mitigated by introducing **inducible promoters** to regulate transcriptional activity.

The team identified two stress-inducible promoters from the **IIT Madras 2008 iGEM collection**: the  **σ³² Heat Shock promoter (BBa\_K086026)** and the  **σ³⁸ Stationary Phase promoter (BBa\_K086030)**  . These allow transcriptional control through environmental conditions—heat shock and stationary-phase signaling, respectively. The σ³⁸ promoter was selected for most laboratory tests due to its IPTG-regulated dual control, while the σ³² promoter was reserved for future bioreactor applications.

Using these promoters, a new modular set of constructs (**Figures 5–9**) was designed to enable interchangeable regulation between riboswitches, degradation genes, and reporter proteins. These improvements aimed to produce a more stable, controllable, and effective biosensor for **RDX detection and degradation**.

![figura](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/2order-3.avif)

**Figure 5**: σ³⁸ Stationary Phase inducible promoter; first Multiple Cloning Site (MCS) includes EcoRI and XbaI restriction sites, RDX-specific riboswitch, *mCherry* reporter sequence; second MCS includes SpeI and PstI restriction sites ending with **BBa\_B0010** terminator.

![figura](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-6-engi.webp)

**Figure 6**: σ³⁸ Stationary Phase inducible promoter; first MCS includes EcoRI and XbaI restriction sites, a standard RBS, ***mCherry*** reporter sequence; second MCS includes SpeI and PstI restriction sites ending with **BBa\_B0010** terminator.

![figura](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-7-engi.webp)

**Figure 7**: First MCS includes EcoRI, NotI, and XbaI restriction sites, followed by ***xplB*** and **xplA** degradation proteins, each regulated by an RDX-specific riboswitch; second MCS includes SpeI, NotI, and PstI restriction sites.

![figura](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-8-engi.webp)

**Figure 8**: Same as **Figure 7**, except that **standard RBS sequences** are used instead of riboswitches.

![figura](https://static.igem.wiki/teams/5905/images/engineeringpage/figures/figura-9-engi.webp)

**Figure 9**: σ³² Heat Shock inducible promoter; MCS includes EcoRI, NotI, XbaI, SpeI, NotI, and PstI restriction sites (standard iGEM prefix and suffix), closed with **BBa\_B0010** terminator.

## **Results**

---

Even though we only received the prototype shown in **Figure 2** (the first order included the sequences of the prototypes shown in **Figures 2–4**), we decided to conduct several preliminary tests to evaluate the functionality of this device. The vector was first resuspended and successfully transformed into electrocompetent *E. coli* DH5α cells. The transformed culture was then inoculated in Luria Broth (LB) to perform a DNA extraction for restriction digestion and to prepare cryopreserved stocks for later use. Two restriction digestion reactions were carried out, one using **NotI** and another using **XbaI** and **SpeI** on another, confirming both the expected restriction sites and the correct prototype size. **Figure 10** shows the 1% agarose gel electrophoresis in 1X TAE (Tris-acetate-EDTA) buffer of the construct depicted in **Figure 2**. The first lane contains the 1 kb DNA ladder, followed by the undigested plasmid with prototype DNA, the NotI digestion, and finally the XbaI and SpeI digestion. The results confirmed the expected banding pattern for the construct.

![](https://static.igem.wiki/teams/5905/resultspage/figure10a.webp)
![Virtual digest](https://static.igem.wiki/teams/5905/resultspage/figura-10b.webp)

**Figure 10**: To the left a 1% agarose in 1X TAE buffer gel electrophoresis of device shown in **Figure 2** and to the right, a virtual digest of the same sequence using benchling.

After experiencing some challenges with the synthesis order, likely due to the constitutive promoter overworking the bacteria, we decided to replace it with an inducible promoter. We subsequently identified the **σ38 Stationary Phase promoter** designed by the **IIT Madras 2008 iGEM team**. This promoter is activated by the stationary-phase sigma factor and includes **Lac operator sites**, allowing it to be repressed by LacI unless IPTG is added to the medium. In other words, it is a **dual-regulated promoter**, responsive to both cellular growth phase and chemical induction. This modification was expected to reduce the stress caused by continuous transcription, potentially resolving the issue of stunted colony growth.

The same iGEM team also developed a **σ32 Heat Shock promoter**, which we plan to use in future experiments involving bench-scale bioreactor testing. However, for most laboratory assays, the stationary-phase promoter will serve as our primary regulatory element.

With this improvement addressing one potential limitation, we also decided to replace the reporter protein. **AmilGFP**, a yellow chromoprotein, proved difficult to visualize in LB medium during early qualitative assays, as its signal was barely distinguishable. To improve detection, we substituted it with ***mCherry***, a red fluorescent protein that offers higher contrast with in the medium. With these two design changes implemented, a **second synthesis order** was placed for the genetic constructs shown in **Figures 5–9**.

After a few weeks, all synthesis orders were unfortunately canceled except for one, the construct described in **Figure 8**. Upon receiving this sequence from IDT, it was transformed into electrocompetent *E. coli* DH5α, cryopreserved with 50% glycerol, and subjected to plasmid extraction using a *ZymoPURE II Plasmid Purification Kit*. The extracted DNA was then analyzed via **restriction digestion** to verify the expected vector and insert sizes. The gel electrophoresis results are shown below: the first well contains the **NEB 1 kb ladder**, followed by the **undigested plasmid**, and lanes three and four show the **NotI** and **XbaI** + **SpeI** digestions, respectively.

![gel electrophoresis](https://static.igem.wiki/teams/5905/resultspage/figure-11a.webp)
![Virtual digest](https://static.igem.wiki/teams/5905/resultspage/figure-11b.webp)

**Figure 11**: To the left a 1% agarose in 1X TAE buffer gel electrophoresis of device shown in **Figure 8** and to the right, a virtual digest of the same sequence using Benchling.

Later, RDX detection tests were conducted using the device shown in **Figure 2**. This construct contains the riboswitch and **amilGFP** as the reporter gene, meaning that any detection of RDX could be qualitatively observed through color change. The bacteria were inoculated in LB medium with different RDX concentrations. However, the results were inconclusive because LB is naturally yellow, which interfered with the visualization of the yellow amilGFP signal.

To address this, we switched to colorless media: **M9, M9 supplemented with glucose** , and **RPMI-1640** . Although RPMI-1640 is typically used for eukaryotic cell culture, we tested it due to its translucency. Three concentrations of RDX were evaluated in these media, and analyses were performed in triplicate for M9 with glucose and RPMI-1640.

After 24 hours of incubation, a slight yellow color was observed, suggesting potential RDX detection and *amilGFP* expression. Quantitative absorbance measurements at **490 nm** were performed (**Table 1**) to validate these preliminary results. The cultures were then incubated for an additional 24 hours to determine whether the signal would intensify. After 48 hours, a stronger yellow color was visible. To better visualize this result, 2 mL bacterial pellets were generated, in which the yellow color was clearly observed. Additional absorbance measurements were also performed to confirm this observation (**Table 2**). Below are the images of the pellets collected for the colorimetric assay.

![figura](https://static.igem.wiki/teams/5905/resultspage/figura12.webp)

**Figure 12**: Bacterial pellets of 1.0 µM RDX solution test on RPMI-1640 medium. From left to right the microtubes are as follows: (1) control, (2) first replicate of test, (3) second replicate of test, (4) third replicate of test.

![figura](https://static.igem.wiki/teams/5905/resultspage/figura13.webp)

**Figure 13**: Bacterial pellets of 2.0 µM RDX solution test on RPMI-1640 medium. From left to right the microtubes are as follows: (1) control, (2) first replicate of test, (3) second replicate of test, (4) third replicate of test.

![figura](https://static.igem.wiki/teams/5905/resultspage/figura14.webp)

**Figure 14**: Bacterial pellets of 3.8 µM RDX solution test on RPMI-1640 medium. From left to right the microtubes are as follows: (1) control, (2) first replicate of test, (3) second replicate of test, (4) third replicate of test.

![figura](https://static.igem.wiki/teams/5905/resultspage/figura15.webp)

**Figure 15**: Bacterial pellets of 1.0, 2.0, and 3.8 µM RDX solution test on M9 medium. From left to right the microtubes are as follows: (1) control - in which no growth was expected because there was no source of carbohydrates, (2) test of 1.0µM of RDX solution, (3) test of 2.0µM of RDX solution, (4) test of 3.8 µM of RDX solution.

![figura](https://static.igem.wiki/teams/5905/resultspage/figura16.webp)

**Figure 16**: Bacterial pellets of 1.0 µM RDX solution test on M9 + 0.2% Glucose medium. From left to right the microtubes are as follows: (1) control, (2) first replicate of test – it is suspected that this tube was not inoculated, (3) second replicate of test, (4) third replicate of test.

![figura](https://static.igem.wiki/teams/5905/resultspage/figura17.webp)

**Figure 17**: Bacterial pellets of 2.0 µM RDX solution test on M9 + 0.2% Glucose medium. From left to right the microtubes are as follows: (1) control, (2) first replicate of test, (3) second replicate of test, (4) third replicate of test.

![figura](https://static.igem.wiki/teams/5905/resultspage/figura18.webp)

**Figure 18**: Bacterial pellets of 3.8 µM RDX solution test on M9 + 0.2% Glucose medium. From left to right the microtubes are as follows: (1) control, (2) first replicate of test, (3) second replicate of test, (4) third replicate of test.

After 48 hours of incubation, visible color differences were observed among the bacterial pellets. In nearly all **+RDX tests**, at least two out of three replicates exhibited a yellow color, except for those cultured in **RPMI-1640 medium**. Notably, none of the **–RDX controls** showed any yellow color, confirming that the observed signal was specific to the RDX-treated samples. Pellets from the **M9 medium** displayed the most pronounced yellow color compared to their controls, suggesting stronger expression of ***amilGFP***. Interestingly, despite this visible intensity, the corresponding absorbance readings were lower than those from RPDMI-1640 and M9+0.2% glucose media.

As shown in the bar charts in **Figures 19–21**, the absorbance readings for M9 medium were consistently lower than those for M9 + glucose and RPMI-1640. The combination of stronger visual pigmentation but lower absorbance may indicate a **lower bacterial growth rate but higher relative reporter protein expression**. These results suggest that *E. coli* grown in the minimal medium without a carbon source may have had **greater permeability to RDX**, leading to enhanced riboswitch activation and, consequently, increased reporter gene expression.

Another important observation is that the **M9 control** showed little to no growth, which was expected for inoculations in a minimal medium lacking a carbon source. This outcome could indicate two possibilities: (1) no cells were transferred to the control tube, resulting in no growth, or (2) the presence of RDX in the treated samples may have **stimulated or supported limited bacterial growth**. In either case, this experiment should be repeated to confirm reproducibility and rule out experimental anomalies. In contrast, the **M9 + glucose** cultures exhibited weaker yellow coloration, which could be due to **reduced RDX permeability** caused by the presence of glucose. However, as expected, these samples showed higher overall absorbance values, reflecting **greater bacterial growth** relative to the other media.

Finally, samples cultured in **RPMI-1640 medium** displayed no detectable yellow pigmentation across all RDX concentrations. Although this medium is **nutrient-rich and optimized for eukaryotic cell culture**, it was selected for testing due to its translucency and personal communication of *E. coli* growth in it. As shown in **Figures 12–14**, no pigmentation was observed in any replicate. The enriched composition of RPMI-1640 potentially interfered with RDX permeability, preventing riboswitch activation and subsequent reporter expression.

## **Measurement Charts**

---

\* These RDX concentrations were chosen based on the interval of RDX detection of the riboswitch as stated by [1]-[2].

**Table 1**: Absorbance Readings at 490 nm after 24 hours of incubation. N/A means there was a failed inoculation.

| Medium | 1 µM | 2 µM | 3.8 µM |
| M9 | 1.058 | 1.268 | 0.593 |
| M9 + 0.2% Glucose | N/A | 1.067 | 1.216 |
| 1.397 | 1.271 | 0.465 |
| 1.360 | 1.414 | 1.279 |
| RPMI-1640 | 2.398 | 0.854 | 2.584 |
| 0.111 | 0.869 | 2.449 |
| 0.008 | 2.093 | 1.533 |

**Table 2**: Absorbance Readings at 490 nm after 48 hours of incubation. N/A means there was a failed inoculation.

| Absorbance Readings at 490 nm; 48 hours | | | |
| Medium | 1 µM | 2 µM | 3.8 µM |
| M9 | 1.699 | 2.243 | 1.384 |
| M9 + 0.2% Glucose | N/A | 2.776 | 3.832 |
| 2.989 | 4.950 | 3.830 |
| 2.637 | 4.970 | 4.518 |
| RPMI-1640 | 2.732 | 2.716 | 2.942 |
| 2.487 | 2.578 | 2.857 |
| 2.590 | 2.931 | 2.615 |

**Table 3**: Absorbance Readings of Controls at 490 nm after 24 and 48 hours of incubation.

| Medium | 24 hours | 48 hours |
| M9 | 0.019 | 0.013 |
| M9 + 0.2% Glucose | 1.373 | 5.030 |
| RPMI-1640 | 0.182 | 2.467 |

**Table 4.** Average, standard deviation and standard error of absorbance measurements of samples inoculated in M9 + 0.2% Glucose with different concentrations of RDX (1µM, 2µM, and 3.8µM).

| Medium | RDX (µM) | Time (h) | Rep1 | Rep2 | Rep3 | Control Mean | Mean | SD | SE |
| M9 + Glucose | 1 | 24 | N/A | 1.067 | 1.216 | 1.373 | 1.142 | 0.105 | 0.075 |
| M9 + Glucose | 1 | 48 | N/A | 2.776 | 3.832 | 5.03 | 3.304 | 0.746 | 0.528 |
| M9 + Glucose | 2 | 24 | 1.067 | 1.271 | 1.414 | 1.373 | 1.251 | 0.174 | 0.101 |
| M9 + Glucose | 2 | 48 | 2.776 | 4.95 | 4.97 | 5.03 | 4.232 | 1.261 | 0.728 |
| M9 + Glucose | 3.8 | 24 | 1.216 | 0.465 | 1.279 | 1.373 | 0.987 | 0.453 | 0.261 |
| M9 + Glucose | 3.8 | 48 | 3.832 | 3.83 | 4.518 | 5.03 | 4.06 | 0.397 | 0.229 |

**Table 5.** Average, standard deviation and standard error of absorbance measurements of samples inoculated in RPMI-1640 with different concentrations of RDX (1µM, 2µM, and 3.8µM)

| Medium | RDX (µM) | Time (h) | Rep1 | Rep2 | Rep3 | Control Mean | Mean | SD | SE |
| RPMI-1640 | 1 | 24 | 2.398 | 0.111 | 0.008 | 0.182 | 0.839 | 1.351 | 0.780 |
| RPMI-1640 | 1 | 48 | 2.732 | 2.487 | 2.59 | 2.467 | 2.603 | 0.123 | 0.071 |
| RPMI-1640 | 2 | 24 | 0.854 | 0.869 | 2.093 | 0.182 | 1.272 | 0.711 | 0.411 |
| RPMI-1640 | 2 | 48 | 2.716 | 2.578 | 2.931 | 2.467 | 2.742 | 0.178 | 0.103 |
| RPMI-1640 | 3.8 | 24 | 2.584 | 2.449 | 1.533 | 0.182 | 2.189 | 0.572 | 0.332 |
| RPMI-1640 | 3.8 | 48 | 2.942 | 2.857 | 2.615 | 2.467 | 2.805 | 0.170 | 0.098 |

![](https://static.igem.wiki/teams/5905/resultspage/figure-19-new.webp)

**Figure 19**: Bar chart illustrating absorbance readings at 490 nm after 24 and 48 hours of incubation for bacterial cultures grown in various media with 1 µM RDX. The average absorbance readings of triplicates from M9 + 0.2% glucose and RPDMI-1640 was calculated and used for this chart.

![](https://static.igem.wiki/teams/5905/resultspage/figure-20-new.webp)

**Figure 20**: Bar chart illustrating absorbance readings at 490 nm after 24 and 48 hours of incubation for bacterial cultures grown in various media with 2 µM RDX. The average absorbance readings of triplicates from M9 + 0.2% glucose and RPDMI-1640 was calculated and used for this chart.

![](https://static.igem.wiki/teams/5905/resultspage/figure-21-new.webp)

**Figure 21**: Bar chart illustrates absorbance readings at 490 nm after 24 and 48 hours of incubation for bacterial cultures grown in various media with 3.8 µM RDX. The average absorbance readings of triplicates from M9 + 0.2% glucose and RPDMI-1640 was calculated and used for this chart.

The most interesting results were obtained from the **M9 medium without glucose**. These samples were originally expected to show **no bacterial growth**, as no carbon source was provided—consistent with the **M9 control**, which showed no growth. Surprisingly, however, *E. coli* inoculated in M9 medium containing **RDX** not only grew but also exhibited the **most intense yellow pigmentation** among all treatments. This observation suggests that the cells may have utilized **RDX** or possibly the **acetone used to dissolve** it as an alternative carbon source.

In future experiments, this assay should be **repeated with a higher number of replicates** for both control and experimental groups to corroborate these findings. If reproducible, subsequent analyses should investigate whether *E. coli* is capable of degrading RDX in the absence of **xplA** and **xplB**, which could indicate previously unreported metabolic activity. Confirming this possibility would have significant implications for the future direction of the project and the design of the biosensor system.

Overall, these findings suggest that the biosensor construct remains **potentially responsive to RDX exposure**, particularly under minimal medium conditions where **membrane permeability may be enhanced**. Nonetheless, **further quantitative validation** and **repeated trials** are required to confirm these preliminary results and assess the reproducibility and sensitivity of the system.

## **Ongoing:**

---

As previously mentioned, the second synthesis order presented a challenge, as only one out of the six ordered constructs were successfully delivered. Upon closer inspection of the submitted sequences, we identified that in both control constructs (**Figures 5** and **6**), a **termination sequence** had been mistakenly placed immediately after the promoter. This likely explains why both constructs encountered issues during transformation.

Additionally, we noticed that the selected vector was not ideal for our construct design. Up to this point, all sequences had been ordered using an **expression vector** (pET-IDT) that already included its own promoter, ribosome binding site (RBS), and terminator elements surrounding the synthesized insert. This configuration may have interfered with proper transcription of our designed sequences, contributing to the synthesis and transformation difficulties reported by IDT.

After identifying these issues, the incorrect placement of the termination sequence in both control constructs was corrected, and a **third synthesis order** was placed for the constructs represented in **Figures 5, 6, 7, and 9**, this time using **IDT’s pUCIDT (Kan)** vector, a much better fit for our genetic devices. As of this writing (Sunday, October 5, 2025), the order has not yet arrived; however, the team has received confirmation from an IDT representative that “the lab has identified perfect colonies for all four genes in the new order.” We expect the sequences to arrive by **October 14, 2025**. Although we will not be able to test these constructs before the **2025 Grand Jamboree**, we plan to continue developing and characterizing them after this year’s competition cycle concludes.

## **References**

---

[1] Eberly, J. O., Mayo, M. L., Carr, M. R., Crocker, F. H., & Indest, K. J. (2019). Detection of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) with a microbial sensor. Journal of General and Applied Microbiology, Advance Publication. https://doi.org/10.2323/jgam.2018.08.001

[2] Mayo, M. L., Eberly, J. O., Crocker, F. H., & Indest, K. J. (2020). Modeling a synthetic aptamer-based riboswitch biosensor sensitive to low hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) concentrations. PLOS ONE, 15(11), e0241664. https://doi.org/10.1371/journal.pone.0241664

[3] iGEM Foundation. (n.d.). Registry of Standard Biological Parts. Retrieved October 5, 2025, from https://parts.igem.org/Main\_Page

{% endblock %}