iGEM 2025 — Team hongkong-jss

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
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![](https://static.igem.wiki/teams/5760/banner/2025-hongkong-jss-wiki-banner-zip-3-1.webp)

## Wet Lab Log

Below is the embedded wet lab log documenting our experimental procedures and results: *(if it does not load please refresh)*

Your browser does not support PDFs. [Download the PDF](https://static.igem.wiki/teams/5760/engineering/engineering/wet-lab-log.pdf).

## Hardware Log

### 27/05/2025 – 10/07/2025: Testing of Bacterial Alginate Beads

Initiated experiments to evaluate the performance of the PbrR-pPbr lead biosensor encapsulated in alginate beads.

* Prepared and optimized alginate–bacteria mixtures with varying sodium alginate and calcium chloride concentrations.
* Conducted bead formation trials using different lead (II) nitrate concentrations (0 mM – 10 mM).
* Observed colour changes as an indicator of lead detection and biosensor responsiveness.
* Refined bead preparation—controlled bead size, viscosity, and hardening rate by adjusting temperature and mixing ratio.
* Established a standardized testing workflow for consistent bead generation and data reproducibility.

### 11/07/2025 – 08/08/2025: Development of fluidic Modules

Focused on designing and testing millifluidic systems to automate bead formation and sampling processes.

* Designed two key modules: Module 1 — Alginate + GM E. coli Mixer and Module 2 — Bead Generator.
* Conducted literature reviews referencing Microfluidic Mixing: A Physics‑Oriented Review (MDPI, 2023) and Fundamentals of Microfluidic Mixing for LNP Synthesis (Inside Therapeutics, 2025) to inform channel geometry and flow strategies.
* Held design meetings to align hardware and fabrication plans; finalized prototype layout and material sourcing.
* Testing of the millifluidic modules prototype.
* Assembled the modular millifluidic body—combining sensors, pumps and electronic controls.

### 09/08/2025 – 10/09/2025: Converging Towards a Portable System

Transitioned from bench-top testing to a compact, deployable prototype.

* Developed and assembled the E. coli culture incubator system with temperature, humidity, and optical density control.
* Installed the UVC sterilization module for post‑use deactivation.
* Integrated the AI vision‑based colour sensor using ESP32‑CAM modules.
* Designed and tested the custom sieve assembly for clear bead visualization.
* Implemented IoT alert functions via SMS and WhatsApp notifications.
* Integrated an LCD touch screen for on‑site display of detection results.
* Developed and connected the website and backend software for live monitoring and system management.
* Tested full system workflow—incubation, detection, data transmission, and alert functions—to ensure stable operation.

### 11/09/2025 – 03/10/2025: Receiving Feedback from Stakeholders and Plan for Future Development

Engaged with experts and users to refine the system and prepare for scaling.

* Collected feedback from academic advisors, farmers, and environmental experts on usability and deployment.
* Evaluated biosafety protocols, waste treatment options, and applications for environmental monitoring.
* Outlined next-phase goals: improving throughput, integrating sensing electronics, and establishing on-site detection capability.
* Planned for future testing with real field water samples and optimized bacterial strains.

## Human Practice Log

### Stage 1 (22/3 - 26/5)

| Time | Stakeholder | Event | Location | Description |
| --- | --- | --- | --- | --- |
| 22/03/2025 | Public | Survey | Online and in-person | We surveyed online and in person to study public perception of medical waste pollution. |
| 31/03/2025 | Industry(medical) | Survey to doctor | Online | We did a survey to doctors to investigate the prevalence, disposal practices, environmental awareness, and perspectives on our target |
| 04/04/2025 | Industry(farming) | Investigation with Pet shop and aquarium | In person, Tung choi st. | We did an investigation on aquarium and pet shop owner along the street and found importance of public education and reinforce our project |
| 22/04/2025 | Government | Interview with AFCD and DSD | Online via email |  |
| 28/04/2025 | Public | Street Interview on Public Awareness of Environmental Issues | Kowloon City district | We conducted a public survey to investigate their awareness and opinions on use of genetically modified bacteria in water testing, how people dispose of expired or leftover medications, and their perceptions of local river pollution |
| 04/05/2025 | Industry (Farming) | Interview with Pig Farm (Yiu Sang pig farm) Owner Mr. Lo Yam-keung | Lung Wo village, Sai Kung | Mr. Lo offered several suggestions about antibiotic use in pig farming, regulations, consumer concerns, and help us define our project's focus point. |
| 10/05/2025 | Industry (Farming) | Interview with Fish Farm Owner Mr Chu Tak-nang | Nam Sang Wai, Yuen Long | Mr. Chu emphasized that antibiotic use in aquaculture varies by species and farming density, and that there are advantages to this. |
| 15/05/2025 | Science/ Academia | Interview with Professor Wong Ming-hung | Hong Kong Baptist University | Professor Wong's insights guided us to improve both the technical clarity and the societal framing, with guided us on the ethical aspect. He also advises on communication and the global context. |
| 17/05/2025 | Science/ Academia | Interview with Professor Yuan Zhiguo | City University of Hong Kong | Mr Yuan expressed the urgency of developing accessible solutions and future development of us to gain publicity and promotion. |
| 26/05/2025 | Science/ Academia | Interview with Doctor Cheung Ka Tik | Online meeting | Dr. Cheung offered several suggestions about combating antibiotic misuse and pollution through regulatory reform, public engagement, technological innovation, and developing the Remedix system for environmental monitoring and clinical diagnostics. |
| 26/05/2025 | Industry(medical) | Interview with Hong Kong Social Enterprise Veterinary Hospital | Mong Kok |  |

### Stage 2 (27/5 - 10/7)

| Time | Stakeholder | Event | Location | Description |
| --- | --- | --- | --- | --- |
| 01/06/2025 | Science/ Academia | Interview with Professor Tam Fung-yee | Hong Kong Metropolitan University | Prof. Tam advised insight into GM E. coli reporter design like speed and sensitivity, testing alginate beads as a containment platform, ensuring clear concentration readouts and warnings for the user to detect pharmaceutical residues like tetracycline and salicylate. |
| 10/06/2025 | Science/ Academia | Interview with Professor Tsui Tsz-ki, Martin | Chinese University of Hong Kong | Professor Tsui offered several suggestions about the scientific and social insight and global applications of our method. |
| 21/06/2025 | Science/ Academia | Interview with Professor Butaye and Miss Sandy Ng | City University of Hong Kong | Prof. Butaye provided both scientific and practical guidance for refining Remedix, underscored the issue and express belief on our project. |
| 29/06/2025 | Industry (Fishing) | Interview with Fish Farm Owner Mr. Hui Chun-yi Johnny | Lau Fau Shan, Yuen Long | Mr Hui provided insight into the challenges faced by fish farmers in managing wastewater, directions for improvement, and belief in our project. |
| 10/7/2025 | Industry (Fishing) | Interview with Fish Farm (Choi Gei Fish Farm) Owner Mr. Law | Sam Mun Tsai, Tai Po | Mr. Law expressed the potential value of Remedix, and suggested our project for accessibility to the public. |

### Stage 3 (11/7 - 10/9)

| Time | Stakeholder | Event | Location | Description |
| --- | --- | --- | --- | --- |
| 28/07/2025 | Science/ Academia | Interview with Professor Frank Lam | Hong Kong University of Science and Technology | Professor Lam's feedback helped us refine both the technical and strategic aspects of our project. |
| 08/08/2025 | Science/ Academia | Interview with Professor Wu Peng | School of public health, Hong Kong University | Professor Wu offered several suggestions about the societal and behavioral dimensions of antibiotic resistance, defining user groups, awareness strategies, practical deployment in educational and community settings, surveillance, responsible prescription practices, and public engagement. |
| 22/08/2025 | Science/ Academia | Interview with Professor Chan Wing-hong on 22/8 (1st Time) | Online | Prof. Chan emphasized methods to better introduce, more reliable detection, and to validate the project and future plans. |
| 25/08/2025 | Science/ Academia | Interview with Professor Chan Wing-hong on 25/8 (2nd Time) | Po Leung Kuk Celine Ho Yam Tong College | Dr. Chan offered several suggestions about strengthening the scientific rigor and addressing the sensitivity and selectivity of the machine, improving detection methods, and positioning our work. |
| 02/09/2025 | Science/ Academia | Interview with Professor Benjamin J Cowling | Hong Kong University | Professor Cowling is encouraging the team to design a biosensing platform that is affordable, effective, and scalable. He also initiative toward addressing public health and antimicrobial resistance. |
| 05/09/2025 | Government | Interview with Mr. MOK Wing-cheong, Ringo (The Director of the Hong Kong Drainage Services Department) | Revenue Tower, Wan Chai | Mr. Mok offered several suggestions about policy-level perspective, technical expertise, pollution data on antibiotics and medical waste, regulatory awareness, data transparency, communication through visualizations, credible lab results, environmental narrative, and refining Remedix into a validated, scalable solution. |
| 10/09/2025 | Science/ Academia | Interview with Professor Chan Ping Lung | Hong Kong Metropolitan University | Professor Chan offered several suggestions about the scientific validity, environmental safety, and practical usability of the Remedix project. |

### Stage 4 (11/9 - 3/10)

| Time | Stakeholder | Event | Location | Description |
| --- | --- | --- | --- | --- |
| 18/09/2025 | Industry (Farming) | Interview with Pig Farm Owner Mr. Tang Yuk-hang, Ken | Deep bay, Lau Fau Shan, Yuen Long | Mr. Tang expressed his belief in our project. |
| 03/10/2025 | Industry(medical) | Interview with Doctor Lau YB And Doctor Leung TK | Online meeting | Dr. Lau YB and Dr. Leung TK's advice helped refine both the technical and social directions of Remedix. |

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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![Banner Image](https://static.igem.wiki/teams/5760/banner/2025-hongkong-jss-wiki-banner-zip-4-1.webp)

##### Project Navigation

* [Introduction](#introduction)
* [Cycle I: Design](#cycle1-design)
* [Cycle I: Build](#cycle1-build)
* [Cycle I: Test](#cycle1-test)
* [Cycle I: Learn](#cycle1-learn)
* [Cycle II: Design](#cycle2-design)
* [Cycle II: Build](#cycle2-build)
* [Cycle II: Test](#cycle2-test)
* [Cycle II: Learn](#cycle2-learn)
* [Cycle III: Design](#cycle3-design)
* [Cycle III: Build](#cycle3-build)
* [Cycle III: Test](#cycle3-test)
* [Cycle III: Learn](#cycle3-learn)
* [Conclusions](#conclusions)
* [Limitations](#limitations)
* [References](#References)

# Engineering Success

During our project experiments, we used engineering principles to organize our work, plan our experiments, and solve problems. We broke the project into smaller, easier-to-handle steps, which let us repeat the engineering process multiple times and improve our methods along the way.

This organized approach was really helpful when our experiments didn't work and we needed to figure out what went wrong. In the next section, we'll show how we successfully completed our project by using engineering design principles and continuously going through cycles of designing, building, testing, learning, and redesigning.

## Cycle I: Design

### Identifying the local problem and solution

At the beginning of our project, our team met to brainstorm local problems that could be solved using synthetic biology. We identified pharmaceutical waste in Hong Kong's water sources as a significant issue. Through background research and interviews with various groups - including industry representatives, citizens, farmers, aquaculture farmers, medical professionals, environmental professionals, and government officials - we confirmed that pharmaceutical wastewater pollution is a real problem in Hong Kong.

While Hong Kong has comprehensive regulations and systems for pharmaceutical waste disposal plus effective wastewater treatment, there are known cases of misuse and regulatory violations by different parties, including ordinary citizens, often due to a lack of awareness or education. This background research emphasized the importance of developing solutions for this problem and using synthetic biology to educate the public about the issue.

Through further research, we discovered that **tetracycline**, an antibiotic commonly used in aquaculture and pig farming, and **salicylate**, a derivative of aspirin (a common painkiller and anti-inflammatory drug), are frequently found contaminants in Hong Kong's water bodies, including the Kai Tak River, where residues of these compounds have been reported. Although tetracycline is found to be in very low concentrations in the ocean and main rivers of Hong Kong and China, it has potential for bioaccumulation. Furthermore, as our research found out, there are people such as aquaculture farmers using tetracycline as antibiotics to treat fish diseases while not following the guidelines and causing point pollution, which creates a problem.

Consequently, our team's project aims to develop methods to both **detect and degrade** these two contaminants using the power of synthetic biology.

(Chui et al., 2020; Chung et al., 2018; Minh et al., 2009; Wilkinson et al., 2022; Leung et al., 2013; Economist Impact, 2024)

### Project Structure

After confirming our project goals, we divided our project into four main components: **Salicylate Biosensor** **Tetracycline Biosensor** **Salicylate Degradation** **Tetracycline Degradation**. We started our experimental work by focusing on designing the two biosensors, which helped us get familiar with lab techniques and routines since we were high school students experiencing a real research project and using advanced synthetic biology techniques for the first time.

### Chromoprotein Selection and Characterization for Biosensor Development

The first focus of our project was developing biosensors with an emphasis on accessibility, making chromoproteins with **visible readouts that don't require special equipment or techniques**, particularly valuable for our applications. To optimize our reporter gene selection, we began our experimental plan with a comprehensive study of chromoprotein candidates.

While our previous teammates successfully used *dTomato* and *amilCP* as reporter genes, we questioned whether these were the best choices for our current goals. Therefore, this year we expanded our characterization to include three additional candidates: *aeBlue* ([BBa\_K864401](https://registry.igem.org/parts/bba-K864401)), *mRFP1e* (engineered version of [BBa\_E1010](https://registry.igem.org/parts/bba-e1010)), and *SYFP2* ([BBa\_K864100](https://registry.igem.org/parts/bba-k864100)).

We designed expression constructs for each chromoprotein, with particular interest in mRFP1e and aeBlue due to their red and blue colors, respectively. These high-contrast colors improve visual detection with the naked eye, making them ideal for accessible biosensor applications. Although our previous *dTomato* and *amilCP* selections also provided red and blue outputs, literature reports indicate that mRFP1e significantly reduces expression burden on host cells. (Bao et al., 2020) This characteristic theoretically promotes both improved growth rates and increased protein expression levels, potentially enhancing biosensor sensitivity.

Additionally, *amilCP* presents practical limitations in experiments, requiring low temperatures (below 30°C) and extended maturation periods (greater than 16 hours) to achieve full color development. In contrast, aeBlue demonstrates more favorable characteristics, requiring less restrictive environmental conditions and achieving color maturation in shorter timeframes according to published reports.

This chromoprotein characterization study served a another purpose in our project development. Since our team recently learned fundamental biotechnology techniques, including pipetting and cell culturing, working with chromoprotein expression constructs provided an excellent training platform. The obvious visual readouts from color changes in transformed cells allowed us to easily assess whether our experimental techniques and designs were working correctly. This approach enabled us to distinguish between technical execution issues and gene design problems before proceeding to our more complex biosensor constructs, where troubleshooting would be significantly more challenging.

### Chromoprotein Expression Construct Design and Co-expression Strategy

We designed expression constructs for mRFP1e, aeBlue, and SYFP2 using standardized iGEM BioBrick parts. Our constructs incorporated a strong constitutive promoter ([BBa\_J23100](https://registry.igem.org/parts/bba-j23100)), a strong ribosome binding site ([BBa\_B0034](https://registry.igem.org/parts/bba-b0034)), and a double terminator ([BBa\_B0015](https://registry.igem.org/parts/bba-b0015)) to ensure strong and constitutive protein expression.

During the design process, we became curious about an intriguing question: what visual outcome would result if host cells co-expressed two different colored chromoproteins? After obtaining permission from our teacher, we decided to investigate this phenomenon experimentally. Consequently, we designed three additional co-expression constructs containing the following combinations: mRFP1e + aeBlue, mRFP1e + SYFP2, and aeBlue + SYFP2.

This co-expression approach introduced us to an important concept in synthetic biology gene design. Initially, we assumed that co-expressing two proteins would require complete, separate expression units for each gene, including individual promoters, ribosome binding sites, and terminators. However, further research revealed a more elegant and effective strategy commonly employed in synthetic biology applications.

We learned that optimal co-expression can be achieved by directly linking the stop codon of the first coding sequence with the start codon of the second sequence, creating a TAATG junction on the coding strand. This design strategy enables simultaneous translation of both proteins from a single transcript, providing better control over the relative expression levels of the two chromoproteins compared to using separate expression cassettes.

By implementing this approach in our three co-expression constructs, we aimed to investigate the resulting color phenotypes while gaining valuable experience with polycistronic expression design principles that are widely used in synthetic biology applications.

## Cycle I: Build

### Cloning Strategy and Chromoprotein Expression Analysis

We ordered our chromoprotein constructs as gene fragments rather than pre-assembled plasmids. Although direct plasmid ordering would have been more convenient, we chose this approach to practice essential laboratory skills and gain experience with experimental design, as mentioned previously. The gene fragments were synthesized by IDT and cloned into pUC19 vectors at the EcoRI restriction site using the HiFi assembly method (we thank NEB for sponsoring iGEM teams with these kits).

### Initial Cloning Challenges and Troubleshooting

Our first cloning attempt was unsuccessful. Following heat shock transformation and ampicillin selection, numerous colonies appeared on the plates, but none displayed the expected chromoprotein coloration. To diagnose this issue, we performed colony PCR using M13 primers to determine whether any colonies contained our genes of interest. The absence of PCR bands indicated that the colonies were likely false positives, resulting from incomplete EcoRI digestion of the pUC19 vector, as suggested by our teacher. Consequently, host cells containing undigested pUC19 retained ampicillin resistance and formed colonies without harboring our constructs.

For our second attempt, we modified the protocol by extending the pUC19 linearization digestion time from 30 minutes to 2 hours and reducing the amount of vector DNA used (sample volume used from 20 µL (from the miniprep) to 10 µL). Since we lacked access to a nanodrop or spectrophotometer, precise DNA concentration measurements were not available. After ~24 hours of incubation, we successfully observed colored colonies, confirming successful chromoprotein expression strain creation.

![Fig 1. LB/Amp plate showing transformation results of mRFP1e, aeBlue, and mRFP1e+aeBlue coexpression transformants. The colored colonies indicating successful expression of the chromoproteins are shown in the diagram.](https://static.igem.wiki/teams/5760/engineering/1.webp)

Fig 1. LB/Amp plate showing transformation results of *mRFP1e*, *aeBlue*, and *mRFP1e+aeBlue* coexpression transformants. The colored colonies indicating successful expression of the chromoproteins are shown in the diagram.

## Cycle I: Test

### SYFP2 Expression Characteristics

Notably, SYFP2 constructs and co-expression strains containing SYFP2 showed no visible coloration, despite literature reports describing yellow-colored SYFP2 colonies. However, examination under blue light revealed bright fluorescence in some colonies, indicating successful SYFP2 expression. The protein exhibited predominantly fluorescent rather than chromogenic properties under our experimental conditions. The co-expression constructs containing SYFP2 showed neither coloration nor fluorescence, but since this was not our primary focus, we did not pursue further investigation into the reason for the failure of expression.

![Fig 2. Transformation result of SYFP2 transformants. The colonies expressing SYFP2 show a bright fluorescence signal under blue light excitation. However, the colony does not show a yellow coloration under the naked eye, indicating the lack of chromogenic property.](https://static.igem.wiki/teams/5760/engineering/2.webp)

Fig 2. Transformation result of SYFP2 transformants. The colonies expressing SYFP2 show a bright fluorescence signal under blue light excitation. However, the colony does not show a yellow coloration under the naked eye, indicating the lack of chromogenic property.

### Comparative Expression Analysis: dTomato vs. mRFP1e

We proceeded to compare dTomato and mRFP1e expression to evaluate whether mRFP1e truly reduces cellular expression burden and enhances growth as reported in the literature. Both constructs utilized identical pUC19 plasmid vectors with the same promoters, ribosome binding sites, and terminators, and were transformed into TOP10 *E. coli* strains under identical conditions.

From the results below, no matter on agar plate or culture medium, the mRFP1e expression cells showed a much deeper red coloration when compared with dTomato after 14 hours of incubation under 37°C (180 rpm for culture). This shows that *mRFP1e* is a more suitable candidate as a reporter gene.

![Fig 3. The comparison of mRFP1e and dTomato's red color chromoprotein property, where mRFP1e shows better coloration than dTomato in both agar plate and medium culture.](https://static.igem.wiki/teams/5760/engineering/3.webp)

Fig 3. The comparison of mRFP1e and dTomato's red color chromoprotein property, where mRFP1e shows better coloration than dTomato in both agar plate and medium culture.

### Unexpected Co-expression Phenotype

An interesting observation emerged from the mRFP1e and aeBlue co-expression experiments. Colonies consistently displayed darker, deeper red coloration compared to mRFP1e-only constructs. Contrary to our expectation of observing purple coloration (red plus blue), the colonies showed no resemblance to aeBlue's characteristic color. This enhanced red phenotype appeared consistently across both agar plates and liquid cultures, with co-expression strains developing deeper, faster coloration within 12 hours compared to single-protein constructs. However, after >20 hours of incubation, the color of mRFP1e and the co-expression construct becomes the same.

![Fig 4. The comparison of mRFP1e and mRFP1e+aeBlue coexpression's chromoprotein property, where the coexpression shows higher intensity in red color within a shorter period of time within 12 hours than the mRFP1e both on agar plate and liquid medium.](https://static.igem.wiki/teams/5760/engineering/4.webp)

Fig 4. The comparison of mRFP1e and mRFP1e+aeBlue coexpression's chromoprotein property, where the coexpression shows higher intensity in red color within a shorter period of time within 12 hours than the mRFP1e both on agar plate and liquid medium.

We hypothesized that co-expressing two proteins might intensify the apparent coloration, leading us to consider this approach for our biosensor reporter system. However, after consulting with our teacher, we learned that co-expression increases cellular metabolic burden. In biosensor applications, this additional workload could potentially overload cells and interfere with their ability to detect target substances. Consequently, we abandoned this approach and did not pursue further investigation into the mechanism underlying enhanced coloration in co-expression systems.

### SDS-PAGE Analysis of mRFP1e and mRFP1e+aeBlue Coexpression

Out of curiosity, we collected cell lysates for further analysis. The gel diagram is shown below.

![Fig 5. SDS-PAGE analysis of chromoprotein expression. Cell lysates from mRFP1e and mRFP1e+aeBlue expressing cultures were separated by SDS-PAGE. An additional band of unknown identity is visible in the coexpression sample compared to controls.](https://static.igem.wiki/teams/5760/engineering/5-2.webp)

Fig 5. SDS-PAGE analysis of chromoprotein expression. Cell lysates from mRFP1e and mRFP1e+aeBlue expressing cultures were separated by SDS-PAGE. An additional band of unknown identity is visible in the coexpression sample compared to controls.

The gel above showed that in the lane containing mRFP1e+aeBlue coexpression cell lysates, an unknown band appeared when compared to the controls, and the band size does not resemble any of the chromoproteins being expressed. Since this was not the focus of our project, we did not investigate it further. We can only speculate that it may be due to incomplete translation termination in the first coding sequence of our coexpression construct, possibly caused by the overlapping start and stop codons at the junction between the first and second coding sequences, resulting in a slightly larger mRFP1e protein product.

## Cycle I: Learn

From the above cycle, we learned several important conclusions:

#### Technical Skills Development

We familiarized ourselves with the techniques used in cloning and synthetic biology, such as construct design. We learned that the most interesting and amazing part of synthetic biology is that a living system can be modified like a machine with each component, which is the genetic parts, being carefully adjusted, and we can make use of living systems to perform amazing things that can benefit human beings greatly.

#### Experimental Complexity

We learned the complexity of living things. Even though the parts' functions are well documented, when we performed the same experiment, the actual experimental conditions greatly affected the performance of the genetic parts, which reminded us that we should be more careful in carrying out such experiments.

#### mRFP1e Performance

We learned that *mRFP1e* is indeed a better reporter gene than *dTomato*, which our team previously used heavily due to its earlier color appearance and deeper color intensity, which makes it a more obvious readout. We decided to use *mRFP1e* as our reporter gene in future construct designs.

#### aeBlue vs amilCP Comparison

*aeBlue*, on the other hand, shows similar expression properties as *amilCP*, which our team previously used. Therefore, we decided to stick with the more familiar *amilCP* instead of *aeBlue* in our later construct designs.

#### A mysterious finding

The coexpression constructs we made just for fun led to a surprising observation. Although we don't have time to investigate it further, it reminds us how scientific discoveries can be spontaneous and unexpected, and that's what makes science fun!

### Transition to Biosensor module and Degradation module Development

Having gained familiarity with these fundamental techniques, we proceeded to the major component of our project: designing biosensors and degradation modules. According to our original plan, we intended to separately test the biosensor inducible module and degradation modules before combining them into an integrated biosensor system with inducible degradation capabilities. This marks the start of our next engineering cycle:

## Cycle II: Design

### First Biosensor Module Design

#### First Tetracycline Biosensor Design ([BBa\_254KMUU6](https://registry.igem.org/parts/bba-254kmuu6))

For the tetracycline detection system, we utilized the well-characterized *TetR/pTet* regulatory system from iGEM parts [BBa\_R0040](https://registry.igem.org/parts/bba-r0040) and [BBa\_C0040](https://registry.igem.org/parts/bba-c0040). This system serves as a reliable foundation for inducible expression applications. Our construct design incorporated a strong constitutive promoter ([BBa\_J23100](https://registry.igem.org/parts/bba-j23100)) to drive TetR expression, paired with medium-strength RBS ([BBa\_B0032](https://registry.igem.org/parts/bba-b0032)). The TetR coding sequence ([BBa\_25ZXMHWA](https://registry.igem.org/parts/bba-25zxm%20hwa)) was codon optimized for *E. coli* expression.

We used the medium-strength RBS strategically to balance TetR expression levels. Since TetR functions as a repressor protein, excessive expression could reduce biosensor sensitivity by over-repressing the reporter system. Conversely, insufficient TetR expression would result in high basal expression levels, causing false-positive results that compromise detection accuracy.

The construct included the *pTet* promoter sequence from [BBa\_R0040](https://registry.igem.org/parts/bba-r0040), followed by *mRFP1e* as the reporter gene. We chose *mRFP1e* based on our previous experimental results demonstrating superior and faster color development under our laboratory conditions. The construct was completed with a double terminator ([BBa\_B0015](https://registry.igem.org/parts/bba-b0015)) to ensure proper transcriptional termination.

![Working Mechanism of the TetR/pTet Operon.](https://static.igem.wiki/teams/5760/engineering/tetr-ptet-operon.webp)

In this mechanism, the TetR repressor protein blocks transcription from the *pTet* promoter under normal conditions. Upon addition of tetracycline, TetR detaches from the DNA, enabling gene expression. This system allows controlled induction of chromoprotein expression as a reporter signal in response to the presence of tetracycline.

#### First Salicylate Biosensor Design and Challenges ([BBa\_25F9SR86](https://registry.igem.org/parts/bba-25f9sr86))

For salicylate detection, we employed the *NahR/pSal* regulatory system using iGEM part [BBa\_J61051](https://registry.igem.org/parts/bba-j61051). Although this part has been frequently used and characterized within the iGEM community, we encountered a significant documentation issue: the exact locations and boundaries of individual components (NahR coding sequence, pSal promoter, and NahR expression elements) within [BBa\_J61051](https://registry.igem.org/parts/bba-j61051) were not clearly defined.

To address this limitation, we attempted to identify and redesign individual components to potentially enhance salicylate-induced sensitivity. In the original [BBa\_J61051](https://registry.igem.org/parts/bba-j61051), the entire NahR expression unit was oriented in the antisense direction. Our first design approach involved reverse complementing this sequence to the sense orientation, facilitating easier handling and reducing potential confusion during construct assembly.

We identified the NahR reading frame using Benchling's translation tool to locate start and stop codons. For NahR expression, we incorporated a strong constitutive promoter ([BBa\_J23100](https://registry.igem.org/parts/bba-j23100)), medium-strength RBS ([BBa\_B0032](https://registry.igem.org/parts/bba-b0032)), and a double terminator (([BBa\_B0015](https://registry.igem.org/parts/bba-b0015))). Our strategy aimed to moderate NahR expression levels, preventing excessive activation that could increase basal pSal promoter activity and reduce biosensor specificity.

![Working Mechanism of the NahR/pSal Operon.](https://static.igem.wiki/teams/5760/engineering/nahr-psal-operon.webp)

Diagram illustrating the NahR/pSal operon system. The nahR gene encodes the NahR regulatory protein, which binds upstream of the *pSal* promoter. In the absence of salicylate, transcription of the target gene is repressed. When salicylate molecules are present, they bind to NahR, activating the regulator and enabling it to bind to *pSal*, thereby initiating transcription of the downstream gene. In the biosensor construct, the system drives the expression of a chromoprotein, serving as a visual reporter signal that indicates the presence of salicylate.

#### Critical Design Decisions and Subsequent Issues

When identifying the *pSal* promoter and NahR binding site regions, we discovered that different iGEM teams had used varying sequence lengths from [BBa\_J61051](https://registry.igem.org/parts/bba-j61051), creating uncertainty about the optimal boundaries. Unfortunately, we made a decision we later regretted: prioritizing construct size minimization, we excluded portions of the sequence that may have been essential for proper function, potentially explaining our first design's failure.

After extracting what we believed to be the NahR binding site and *pSal* promoter region, we added strong RBS ([BBa\_B0034](https://registry.igem.org/parts/bba-b0034)), *amilCP* ([BBa\_K592009](https://registry.igem.org/parts/bba-k592009)) as the chromoprotein reporter gene, and double terminator ([BBa\_B0015](https://registry.igem.org/parts/bba-b0015)). We subsequently realized a significant design flaw: using identical terminators for both genes within a single construct. This decision likely contributed to the obstacles and delays we encountered during gene synthesis, as identical sequences can cause synthesis and assembly complications.

### Degradation Module Design

#### Tetracycline Degradation Construct ([BBa\_25CS26NB](https://registry.igem.org/parts/bba-25cs26nb))

Through literature review, we identified TetX, a flavin-dependent monooxygenase, as an effective enzyme for tetracycline degradation. TetX breaks down tetracycline into 2-acetyl-2-decarboxamido-tetracycline, a non-toxic, non-antibiotic final product. This single-enzyme solution significantly simplified our experimental design compared to multi-step degradation pathways.

Literature reports confirmed that TetX can be successfully expressed, properly folded, and functionally active in *E. coli* cells, enhancing bacterial tetracycline resistance. Additionally, since tetracycline naturally enters *E. coli* cells (which contributes to its antibiotic mechanism), the TetX enzyme does not require secretion to effectively degrade extracellular tetracycline. (Yang, Moore, Koteva, Bareich, Hughes, & Wright, 2004)

Our construct design incorporated a strong constitutive promoter ([BBa\_J23100](https://registry.igem.org/parts/bba-j23100)) and strong RBS ([BBa\_B0034](https://registry.igem.org/parts/bba-b0034)) to maximize enzyme expression levels and enhance degradation efficiency. We also codon optimized the *TetX* coding sequence ([BBa\_25ZXMHWA](https://registry.igem.org/parts/bba-25zxmhwa)) for *E. coli* expression. We later discovered that *pET* protein expression systems would have provided even stronger expression in *E. coli*, but this insight came too late in our design process to implement.

#### Salicylate Degradation Construct Design ([BBa\_257IE4RA](https://registry.igem.org/parts/bba-257ie4ra))

Salicylate degradation required a more complex approach due to the toxicity of intermediate products. NahG enzyme converts salicylate into catechol, and since salicylate can enter *E. coli* cells naturally, no secretory tags were necessary. However, catechol itself is ecotoxic, meaning that simply degrading salicylate would solve one environmental problem while creating another.

To address this issue, we incorporated XylE enzyme, which converts catechol into non-toxic metabolites according to literature reports. This two-enzyme system required co-expression strategies, building upon our experience with chromoprotein co-expression experiments.

We designed two different co-expression approaches.

The first design used a strong constitutive promoter ([BBa\_J23100](https://registry.igem.org/parts/bba-j23100)) and two RBSs to drive the expression of both *NahG* and *XylE* coding sequences. Since we know that the intermediate product catechol is toxic waste, we don't want it to accumulate in the system, so we designed the expression of XylE to be stronger than that of NahG, ensuring that the breakdown rate of catechol by XylE would exceed its formation by NahG. To achieve this, we used a medium-strength RBS ([BBa\_B0032](https://registry.igem.org/parts/bba-b0032)) for *NahG* and a strong RBS ([BBa\_B0034](https://registry.igem.org/parts/bba-b0034)) for *XylE*, with a double terminator ([BBa\_B0015](https://registry.igem.org/parts/bba-b0015)) added at the end.

For the second approach, we conducted additional research and discovered the possibility of creating fusion proteins using linker sequences. This strategy would produce a single large protein containing both enzyme subunits within one polypeptide chain. Intrigued by this concept and curious about its feasibility for our application, we designed a construct to test this fusion protein approach alongside our traditional co-expression method. However, this design could not be synthesized by any company that we approached, so we could not make any progress with this design.

![Enzymatic Pathway of Salicylate Degradation by NahG and XylE](https://static.igem.wiki/teams/5760/engineering/sal-degrade.webp)

Diagram illustrating the salicylate degradation pathway mediated by NahG and XylE enzymes. In this pathway, NahG (salicylate hydroxylase) converts salicylate into catechol. Catechol acts as a toxic intermediate that can accumulate if not further degraded. To prevent toxicity, XylE (catechol 2,3-dioxygenase) cleaves the aromatic ring of catechol to form 2-hydroxymuconic semialdehyde (HMS), a less toxic and more stable compound.

## Cycle II: Build

### Plasmid Synthesis Difficulties

We initially ordered our constructs as plasmids from IDT in pIDT-Amp vectors. Our bulk order included all the biosensors and degradation constructs. However, all of the degradation constructs failed to be synthesized, forcing us to immediately modify our design sequences, optimize them, and place a new order with Twist Bioscience. In the meantime, we could only focus on testing our biosensor constructs.

## Cycle II: Test

### Establishing a Concentration for Tet Functional Study

Since tetracycline is an antibiotic that inhibits and kills *E. coli*, our host, we need to know which concentration is suitable for the induction of our biosensor while not killing our cells. Actually, we thought about using a tetracycline-resistant strain, but we found out that the resistance mechanism in *E. coli* was actually due to an active pump which pumps out tetracycline from inside the cells, so this approach would definitely interfere with our biosensor which relies on tetracycline inside our cells to bind to the TetR protein.

Therefore, we carried out a growth study with different concentrations of tetracycline. At that time, since we did not have access to a spectrophotometer, we could not measure the OD, but we judged the growth of the cells by the cloudiness of the culture. We carried out a serial dilution of tetracycline with a concentration range of ~600 ng/ml to ~38 ng/ml. Although the MIC of *E. coli* was reported to be 10 μg/ml, we found that at ~300 ng/ml, the growth of the cells was significantly affected after 14 hours, as shown in the diagram below, where the growth only shows after 22 hours, so it is not suitable for our experiment. Therefore, we decided that 200 ng/ml would be the highest concentration for induction.

![Fig 6. Control <i>E. coli</i> cells cultured in different concentrations of tetracycline. In ~600 ng/ml and ~300 ng/ml concentrations, reduced growth in the medium can be observed obviously after 14 hours.](https://static.igem.wiki/teams/5760/engineering/5.webp)

Fig 6. Control *E. coli* cells cultured in different concentrations of tetracycline. In ~600 ng/ml and ~300 ng/ml concentrations, reduced growth in the medium can be observed obviously after 14 hours.

### First Functional Testing Trial on Biosensors

After receiving our first batch of plasmids, we transformed plasmids with biosensor constructs - Tetracycline Biosensor ([BBa\_254KMUU6](https://registry.igem.org/parts/bba-254kmuu6)) and Salicylate Biosensor ([BBa\_25F9SR86](https://registry.igem.org/parts/bba-25f9sr86)) into *E. coli* TOP10 strains and performed ampicillin selection. We obtained colonies and proceeded with functional testing using the following methods:

Individual colonies were inoculated into 10 mL LB/Amp starter cultures and grown for 4 hours at 37°C with 180 rpm shaking. We then transferred 100 μL of starter culture into 10 mL fresh LB/Amp medium containing the respective inducers. For tetracycline testing, we tried concentrations within the normal induction range of 1 ng/mL to 200 ng/mL. For salicylate, we tried concentrations from 0.1 mM to 2 mM.

We monitored color changes at 6, 16, 24, and up to 36 hours post-induction. At each time point, we collected 1 mL of culture and centrifuged at 8000 g for 1 minute to form cell pellets for better color observation, comparing induced cultures to uninduced controls and normal cells with inducer.

Unfortunately, no observable changes occurred in any biosensor setup. We attempted troubleshooting by testing various conditions, including different inducer concentration ranges, extended incubation times, reduced incubation temperatures, and alternative cell strains such as BL21. Despite these modifications, we observed no biosensor response, leading us to conclude that the issue was more likely originated from our construct design, necessitating design revisions.

## Cycle II: Learn

This concludes this engineering cycle that focused on the design, build, and testing of our biosensor designs, and we gained experience that:

#### Technology Awareness

We were amazed at the advances in technology nowadays, where a blueprint of life - DNA - can be synthesized at such low cost and obtained so conveniently, just by clicking online. However, this also made us ignore the importance of planning ahead and our awareness of preparing for sudden changes. Since we were too confident that plasmids could be obtained very easily, when they couldn't be synthesized and our initial plan was interrupted and delayed, we were in a mess and panicked. This taught us a lesson that we should do better preparation for everything.

#### Synthesis Feasibility

We also learned that when designing DNA constructs, another point to consider is the feasibility of synthesizing such DNA. Although it may work biologically, the problem may arise at the synthesis stage.

#### Systematic Problem-Solving

We learned how to approach problem-solving step by step. We modified each component of the experimental conditions one at a time and tested to see if that particular component was causing the experiment to fail. This made us realize how to carry out experiments systematically, and that's the spirit of engineering.

To conclude from our experimental results of functional studies on our biosensors:

#### Biosensor Design Evaluation

Our initial biosensor design seems flawed based on our experimental results. We made significant efforts trying different experimental conditions but still achieved no positive outcomes. However, we are grateful that this experience taught us even more about gene expression in cells and how environmental factors can contribute to a living cell's gene expression.

Therefore, in the next cycle, we will redesign and test the biosensor constructs using the experience we learned in this cycle. Meanwhile, we brainstormed and consulted different experts, such as our teachers, and we hypothesized some reasons for the failure in our biosensor designs, which we will incorporate as corrections in our next cycle.

## Cycle III: Design

### New Tetracycline Biosensor Design Modifications and Rationale ([BBa\_252ZMUOK](https://registry.igem.org/parts/bba-252zmuok))

For our new tetracycline biosensor design, we suspected that the previous construct's failure was due to the strong promoter that we used to drive TetR expression, which is a repressor of *pTet* transcription. Especially when we were using the vector pIDT-AMP, which is a high-copy plasmid, it causes a very high level of transcription. Therefore, it may cause the TetR repression effect to be too strong. Meanwhile, in the tetracycline functional study, we cannot increase the tetracycline concentration due to its inhibitory effect on *E. coli* growth, so the induction concentration must be kept low. Therefore, in the next design, we changed the promoter to a medium-strength constitutive promoter ([BBa\_J23111](https://registry.igem.org/parts/bba-j23111)), hoping this will solve the problem.

Also, we changed the reporter gene from *mRFP1e* to *amilCP* to align with our salicylate biosensor design. This modification was particularly advantageous because amilCP folds more efficiently at lower culturing temperatures (below 30 °C). This enables future experiments where both biosensor types can be cultured together in the same incubator. Moreover, it reflects real-world conditions, as environmental temperatures—especially in aquatic habitats—are typically below 30 °C.

### New Salicylate Biosensor Design Modifications and Rationale ([BBa\_25WAI7EY](https://registry.igem.org/parts/bba-25wai7ey))

For our revised salicylate biosensor design, we hypothesized that the previous failure stemmed from altering the original sequence orientation. In the original BioBrick part, the NahR expression unit was positioned in an antisense orientation, but our initial design modified this to the sense direction. This change may have caused transcriptional interference between the NahR expression machinery and the reporter gene unit, ultimately leading to our first design's failure. To address this issue, we reverted the NahR expression module back to its original antisense orientation while keeping all other design elements unchanged.

## Cycle III: Build

### Second Synthesis Attempt and Extended Delays

For our second trial, we ordered redesigned biosensor constructs and degradation modules from Twist Bioscience as plasmids in pTwist-Amp vectors. Unfortunately, the delivery time far exceeded our expectations. Despite ordering in July, the constructs only arrived in late September. Moreover, synthesis success was limited: only the redesigned *NahR/pSal* salicylate biosensor and *TetX* tetracycline degradation constructs could be successfully synthesized, while all others failed.

### Lessons Learned and Budget Constraints

This experience taught us that while ordering plasmids directly initially seemed more efficient and cost-effective, we had underestimated the difficulties and time costs associated with plasmid synthesis. Significant project time was lost waiting for deliveries or discovering synthesis failures after extended waiting periods.

Upon learning that several constructs could not be synthesized, we immediately placed orders with BGI company. However, budget limitations forced us to order plasmids individually rather than in bulk, causing additional delays. The same synthesis problems recurred, with many plasmids failing to be successfully cloned.

This experience highlighted the importance of better planning and preparation for such contingencies in future projects. We finally received functional constructs in October, significantly impacting our project timeline.

### Initial Construct Validation

For the biosensor modules, the redesigned construct was ordered from Twist Bioscience in pTwist-AMP vector and transformed into both TOP10 and BL21 *E. coli* host strains. Following ampicillin selection, we obtained four colonies and proceeded to functional studies.

For the degradation modules, after ordering from three different companies, we finally obtained the *TetX* and *NahG/XylE* degradation constructs. However, the fusion protein design could not be synthesized by any company. Additionally, due to technical difficulties, the *NahG/XylE* construct could only be synthesized in an intermediate plasmid (pKMV) containing a kanamycin resistance gene, requiring all downstream processing to be performed with kanamycin selection rather than ampicillin.

Both degradation constructs were transformed into *E. coli* TOP10 and BL21 cells and selected on ampicillin or kanamycin LB agar plates respectively.

## Cycle III: Test

### Second Biosensor Trial Results

#### Tetracycline Biosensor Functional Assay

This time, our redesigned construct ([BBa\_252ZMUOK](https://registry.igem.org/parts/bba-252zmuok)) performed as expected. Cultures treated with 100 ng/mL tetracycline (final concentration) began showing blue coloration from amilCP reporter protein expression. When the tetracycline concentration was increased to 200 ng/mL, color intensity correspondingly increased, while control cells showed no significant blue coloration. The blue coloration was clearly visible in the culture medium and became even more pronounced after centrifugation when cells were pelleted.

However, at 10 ng/mL tetracycline, no significant color difference was observed compared to the uninduced control, indicating a detection threshold between 10 and 100 ng/mL under our experimental conditions. The results are shown in the diagram below.

![Fig 7. Our <i>TetR/pTet</i> tetracycline biosensor successfully detected the presence of tetracycline (Tc) by showing a color change in concentrations of 100 ng/mL and 200 ng/mL of Tc.](https://static.igem.wiki/teams/5760/engineering/6.webp)

Fig 7. Our *TetR/pTet* tetracycline biosensor successfully detected the presence of tetracycline (Tc) by showing a color change in concentrations of 100 ng/mL and 200 ng/mL of Tc.

Learning from the above results, we decided to use smaller concentration intervals to better observe the response of our biosensor. The results are shown below.

![Fig 8. Another set of replicates using smaller intervals of tetracycline concentrations to assess the response of our biosensor.](https://static.igem.wiki/teams/5760/engineering/8-2.webp)

Fig 8. Another set of replicates using smaller intervals of tetracycline concentrations to assess the response of our biosensor.

From these results, we can see that the color intensity of this set does not resemble the first set, even though the growth conditions of the two experiments were the same. The difference was that we used overnight culture of the biosensor and diluted it in fresh LB/Amp directly instead of preparing a new starter culture, as we did not have sufficient time for the experiment (wiki freeze was approaching in two days). However, this result taught us that optimizing cell conditions is critical for biosensor performance, and we should carefully control the growth conditions of the cells.

Another noteworthy observation is that we found a mild leaky expression problem, since the 0 ng/mL biosensor pellet shows a noticeable color difference compared to the pUC19 empty vector control. This indicates that we need to better modify and adjust the gene regulation and biosensor conditions.

Nevertheless, we can still observe differences in color intensity between tetracycline-treated biosensors and the controls. This set of results further validates the functionality of our biosensor and proves our design concept.

#### SDS-PAGE validation of the expression

We also performed SDS-PAGE analysis to validate protein expression in our constructs. As shown in the gel image below, the lane containing *TetR/pTet* expressing cells displays a prominent band at approximately 23.3 kDa. However, we must consider that amilCP has a molecular weight of ~25.6 kDa (visible in the amilCP expressing cell lane), which means any leaky expression of amilCP could potentially overlap and interfere with our results.

Despite this potential complication, our functional studies successfully demonstrated that the construct can detect tetracycline. Additionally, when comparing the gel bands, the *TetR/pTet* lane shows a thicker band positioned lower than the amilCP control, indicating a smaller protein size relative to amilCP. Based on these observations, we conclude that the observed band primarily represents TetR expression rather than amilCP leakage.

![Fig 9. SDS-PAGE showing the protein expression in our constructs. In the lane of <i>TetR/pTet</i> expression cells' proteins, an obvious thickened band can be observed at the position of ~23 kDa which matches the size of TetR protein.](https://static.igem.wiki/teams/5760/engineering/7.webp)

Fig 9. SDS-PAGE showing the protein expression in our constructs. In the lane of *TetR/pTet* expression cells' proteins, an obvious thickened band can be observed at the position of ~23 kDa which matches the size of TetR protein.

#### Bead-Based Biosensor Application

As we proposed, using alginate beads to immobilize the biosensor would be an effective way to utilize the biosensors, since this approach reduces the risk of culture spillage, improves viability, enables easier observation, and better maintains the environment for the biosensor cells. (Massana Roquero & Katz, 2022) After confirming the functionality of the biosensors, we tested them in alginate beads. The results are shown below.

![Fig 10. Alginate bead-based tetracycline biosensor response. Beads containing biosensor cells or pUC19 control were incubated with varying tetracycline concentrations (0-200 ng/mL) for 16 hours. Visible color changes indicate successful tetracycline detection in the bead format.](https://static.igem.wiki/teams/5760/engineering/beads2.avif)

Fig 10. Alginate bead-based tetracycline biosensor response. Beads containing biosensor cells or pUC19 control were incubated with varying tetracycline concentrations (0-200 ng/mL) for 16 hours. Visible color changes indicate successful tetracycline detection in the bead format.

As shown in the figure, a color change distinguishable by the naked eye can be observed in beads exposed to 100 ng/mL and 200 ng/mL tetracycline culture medium when compared with pUC19 empty vector control and no Tc added control after 16 hours. This again provides direct evidence that our concept of using beads for biosensor applications is viable.

#### Salicylate Biosensor Functional Assay

We proceeded with functional testing on the new design ([BBa\_25WAI7EY](https://registry.igem.org/parts/bba-25wai7ey)) using the following protocol: selected colonies were incubated in LB/Amp starter culture for 2 hours, then transferred to fresh LB/Amp medium at 1:100 dilution and incubated for an additional 4 hours. We added 100 mM sodium salicylate solution to achieve a final concentration of 1 mM in the culture medium.

Despite multiple attempts and monitoring at 6, 12, and 18 hours post-induction, we observed no significant color changes in our biosensor cultures. This result indicated that our redesigned construct, while showing improved basal reporter expression, still lacked functional salicylate responsiveness. The persistent lack of an inducible response suggested that additional design modifications would be necessary to achieve a working salicylate biosensor.

We then immediately designed a third version, suspecting that the earlier failures were due to truncating part of the *pSal* promoter or the NahR regulatory sites from the original [BBa\_J61051](https://registry.igem.org/parts/bba-j61051) part. Therefore, in the third design, we included the complete sequence of [BBa\_J61051](https://registry.igem.org/parts/bba-j61051) from just before the start codon of the NahR coding sequence until the last base of the part. This may include the intrinsic Pr promoter sequence and RBS, so we added the [BBa\_J23100](https://registry.igem.org/parts/bba-j23100) promoter and [BBa\_B0034](https://registry.igem.org/parts/bba-b0034) RBS upstream of these elements to drive stronger NahR expression. However, at the time of writing this report (with only a few days remaining before the wiki freeze), the plasmid had not yet arrived, forcing us to abandon testing of this design.

### First Trial of Degradation Constructs

#### Tetracycline Degradation Assay

For tetracycline degradation testing, as we learned that TetX can also improve survivability, we decided to try with a higher concentration of tetracycline up to 10 μg/ml (The MIC of *E. coli*), as also suggested by our AI helper. Since this can help us to easily distinguish if our cells have successfully expressed TetX from control cells, the growth difference can be significant. Thus, a 5 μg/ml and 10 μg/ml concentration of tetracycline were used to see the performance of our tetracycline degradation cells.

We found that tetracycline could be detected at an absorbance of 360 nm using a standard spectrophotometer, which allowed us to generate a standard curve with tetracycline solutions prepared in LB medium. (Ivantsova et al., 2025) Since our lab does not have a spectrophotometer, we are grateful to our chemistry teachers for lending us their lab’s instrument for this experiment.

#### Tetracycline standard curve ABS360

We plotted a standard curve of absorbance at 360 nm against standard tetracycline solutions in the range of 1 μg/ml to 10 μg/ml. LB/Amp solution was used as the solvent and blank solution. The standard curve and the linear equation are shown below. The R² of the curve is 0.999 which shows that it's highly reliable.This standard curve proved that this method of measurement is valid and it can reflect the concentration of tetracycline in the culture medium.

![Fig 11. Standard curve of measuring tetracycline concentration in LB/Amp broth at 360 nm with a spectrophotometer.](https://static.igem.wiki/teams/5760/engineering/11-2.webp)

Fig 11. Standard curve of measuring tetracycline concentration in LB/Amp broth at 360 nm with a spectrophotometer.

#### Degradation Protocol and Results

Transformant colonies were selected from ampicillin plates and grown in LB/Amp medium for 2 hours. We then transferred 100 μL starter culture to 10 mL degradation culture and incubated for 4-6 hours until log phase. Tetracycline was added to a final concentration of ~5 μg/mL and ~10 μg/mL, and we immediately measured baseline absorbance (time = 0) by centrifuging cells at 12,000 g for 5 minutes and measuring supernatant absorbance at 360 nm. Subsequent measurements were taken at 2 and 4 hours post-addition.

![Fig 12. Graphs showing the degradation results of tetracycline by our TetX expressing <i>E. coli</i> in 5 μg/mL (a) and 10 μg/mL (b) tetracycline initial concentration after 2 and 4 hours.](https://static.igem.wiki/teams/5760/engineering/degra.webp)

Fig 12. Graphs showing the degradation results of tetracycline by our TetX expressing *E. coli* in 5 μg/mL (a) and 10 μg/mL (b) tetracycline initial concentration after 2 and 4 hours.

Results showed that cells expressing TetX significantly reduced absorbance at 360 nm compared to normal *E. coli* containing pUC19 after 2 hours, indicating decreased tetracycline concentrations in the medium. Additionally, TetX-expressing cells showed larger cell pellet sizes and higher OD600 values compared to pUC19 controls, further supporting our hypothesis of enhanced survival and growth, as shown in the diagram below. However, at the 4 hour time point, the value has not shown further change when compared to the 2 hour time point. This makes us wonder why. We hypothesized that it is due to the decrease of tetracycline to a very low concentration which hinders the enzymatic reaction rate making it slow, and also the *E. coli* cells may have reached a post-log phase, where they are not in a healthy status, so their energy and condition are no longer optimal for the enzymes to work.

Given these results, we subsequently conducted measurements within a 2-hour timeframe to optimize monitoring of tetracycline degradation by our cells. Measurements were taken at 30-minute intervals to better track the degradation activity during the period of highest enzymatic activity.

![Fig 13. Tetracycline degradation assay over 2 hours. Three conditions were tested: LB only, pUC19 control + tetracycline, and TetX-expressing cells + tetracycline. Error bars represent SD of two biological replicates (n=2).](https://static.igem.wiki/teams/5760/engineering/degra2.avif)

Fig 13. Tetracycline degradation assay over 2 hours. Three conditions were tested: LB only, pUC19 control + tetracycline, and TetX-expressing cells + tetracycline. Error bars represent SD of two biological replicates (n=2).

The results graph shown above demonstrates that the tetracycline concentration decreases continuously at 30-minute intervals throughout the measured timeframe. This data further confirms the functionality of our engineered cells and enhances our understanding of their tetracycline degradation kinetics.

In our results, we observed considerable variation in absorbance values—including the initial readings and replicates—despite careful attention to technique. We suspect this variability arises from interference by cells and other components in the LB medium. The reported ABS360 method for tetracycline quantification was developed using pure water, which differs from our experimental conditions. The complex composition of the LB/Amp medium and the presence of cells likely make this measurement less stable. Nevertheless, the clear decrease in absorbance for TetX-expressing strains, compared to the relatively stable control, strongly supports the effectiveness of our degradation cells.

#### Protein Expression Validation

The TetX expression was also confirmed by SDS-PAGE. The expected ~44 kDa TetX band matched the size.

![Fig 14. SDS-PAGE showing the protein expression in our constructs. In the lane of TetX expression cells' proteins, an obvious thickened band can be observed at the position of ~44 kDa which matches the size of the TetX protein.](https://static.igem.wiki/teams/5760/engineering/10.webp)

Fig 14. SDS-PAGE showing the protein expression in our constructs. In the lane of TetX expression cells' proteins, an obvious thickened band can be observed at the position of ~44 kDa which matches the size of the TetX protein.

#### Salicylate Degradation Assay Challenges

Experimental Limitations

For salicylate degradation testing, we planned to add salicylate to a final concentration of 0.5 mM to cultures containing the *NahG/XylE* co-expression construct to study the degradation effect. However, we faced significant limitations due to lack of sophisticated equipment such as UV-spectrophotometers with 230nm wavelength needed to measure concentrations of salicylate, catechol, or the final product (2-hydroxymuconic semialdehyde).

#### Alternative Detection Method

Research revealed that FeCl₃·6H₂O reacts with salicylate to form purple compounds quantifiable by measuring absorbance at 530 nm using our conventional spectrophotometer. We attempted to construct a standard curve using FeCl₃·6H₂O and standard salicylate solutions in LB medium.

#### Technical Difficulties

Unfortunately, we were unable to construct a reliable standard curve even we tried several times. We suspected that our FeCl₃·6H₂O stock had expired, as literature indicated that this chemical forms Fe³⁺ hydroxide over time, preventing accurate standard solution preparation. Due to time constraints, we were forced to abandon this approach for salicylate degradation quantification.

#### Protein Expression Validation

Nevertheless, we also studied if the NahG and XylE proteins were successfully expressed by our construct design. In the protein gel below, NahG (~49.3 kDa) and XylE (~35.1 kDa) matching size bands were observed in the gel photo, which indicates the successful expression.

Importantly, the band corresponding to XylE shows noticeably higher intensity than that of NahG. This confirms our design concept: XylE is expressed at a higher level than NahG, which could result in greater XylE activity within cells carrying this construct and help prevent catechol accumulation during salicylate degradation.

![Fig 15. SDS-PAGE showing the protein expression in our constructs. In the lane of <i>NahG/XylE</i> expression cells' proteins, two thickened bands can be observed at the positions matching NahG (~49.3 kDa) and XylE (~35.1 kDa).](https://static.igem.wiki/teams/5760/engineering/11.webp)

Fig 15. SDS-PAGE showing the protein expression in our constructs. In the lane of NahG/XylE expression cells' proteins, two thickened bands can be observed at the positions matching NahG (~49.3 kDa) and XylE (~35.1 kDa).

## Cycle III: Learn

### Summary

This concludes our project. Through the experimental engineering cycles, we gained valuable insights into our work. We learned that employing an effective and systematic problem-solving approach was essential to successfully cloning our constructs and conducting investigations on our designs. Each iteration of the cycle allowed us to refine our methods, troubleshoot issues, and adapt our experiments based on the challenges we encountered, ultimately leading to a deeper understanding of both the engineering process and the functionality of our biosensors. Particularly due to our inexperience and inadequate planning, we were fortunate that we could obtain experimental results just a few days before the wiki freeze. This experience emphasizes the importance of better planning and preparation.

From our experimental results, we can conclude the following for our project:

#### Validation of Chromoprotein Expression

We successfully validated the expression of the chromoproteins mRFP1e, SYFP2, and aeBlue, as well as mRFP1e/aeBlue coexpression, and compared their expression with the chromoproteins we had available.

#### Proved Tetracycline Biosensor Functionality

Our tetracycline biosensor ([BBa\_252ZMUOK](https://registry.igem.org/parts/bba-252zmuok)) can detect the presence of tetracycline and express chromoprotein as a reporter gene. The expression of the chromoprotein produced a visible signal that could be observed with the naked eye, without requiring specialized equipment or techniques.

#### Tetracycline Biosensor Shown Concentration-Dependent Response

We demonstrated that the tetracycline biosensor can reflect the concentration of the target by showing differences in color intensity of the chromoprotein reporter gene.

#### Proof of concept - The biosensor is functional in bead design

Fixing into alginate beads is a common way of immobilizing biosensors or GM cells, which makes them more viable, easier to see, and can better maintain the environment of the biosensor. As we proposed, we proved that the biosensor is functional in the bead format, which greatly enhances the practical value of the biosensor in real-world settings.

#### Shown Tetracycline Degradation Capability

We also successfully developed a tetracycline degradation strain ([BBa\_25CS26NB](https://registry.igem.org/parts/bba-25cs26nb)) that can effectively degrade tetracycline, making our concept of creating a bioremediation solution to address tetracycline contamination in water feasible.

#### Confirmed Differential Expression of NahG and XylE

We successfully designed and expressed the NahG/XylE salicylate degradation construct ([BBa\_257IE4RA](https://registry.igem.org/parts/bba-257ie4ra)).
Although its activity could not be directly tested, SDS-PAGE confirmed the expression
of both proteins, with XylE showing higher levels than NahG. This result supports
our design principle that increased XylE activity helps prevent toxic catechol buildup
during salicylate degradation, and demonstrates how protein expression levels can be finely
tuned through thoughtful selection of genetic components—highlighting once again the remarkable
potential of synthetic biology.

#### Lessons from DNA Ordering

Better planning and design in DNA synthesis are needed in the future. Due to insufficient awareness in DNA design, many of our designs could not be synthesized, which nearly caused our project to fail completely.

With these results and conclusions, we have successfully demonstrated that our concept is viable and proven that our tetracycline biosensor and degradation designs can function as intended. However, we are fully aware that significant room for improvement exists and that further investigations are necessary to refine the system.

### Limitations and Areas for Improvement

Several major considerations need to be addressed in our project:

#### Incomplete Salicylate Module

Regarding salicylate, we were unable to successfully create the biosensor and did not have sufficient time to test our salicylate degradation cells. This leaves a gap to address in future work toward our project goals.

#### Enhancing Sensitivity to Low Concentrations

Sensitivity is crucial for a biosensor. We would like to explore whether the sensitivity of the biosensor can be maximized to make it more applicable in various situations.

#### Integration of Detection and Degradation Modules

We would like to integrate the *TetX* module with the biosensor module, enabling TetX expression to be induced by the presence of tetracycline. This would create a more sophisticated system of conditional TetX expression when needed by our cells.

#### Minimizing Leaky Expression in Biosensors

A common issue is that reporter genes may exhibit leaky expression due to culturing conditions, such as prolonged incubation. In the tetracycline biosensor, mild leaky expression is observed, so further modifications for tighter control are needed.

#### Single-Cell Integration

We are interested in determining if the detection and degradation of tetracycline can be integrated into a single cell.

#### Alginate Bead Integration for Real-World Applications

As our hardware team developed a machine to automate the process of making cells into beads, we needed time to test compatibility and determine if detection and degradation efficiency are maintained in the alginate beads.

#### Ensuring Safety Measures

Safety is critical to our project. We need to implement measures such as kill switches or containment mechanisms when working with engineered organisms. Additionally, exploring cell-free expression systems could help mitigate concerns regarding the use of genetically modified organisms.

Therefore, we are beginning a new round of the engineering cycle to address these important points. By combining our biological system with the device, we aim to improve our product's functionality, applicability, and safety. It is crucial for us to thoroughly investigate every aspect to ensure that our system meets the necessary standards and regulations for real-world implementation.

![Thank you for watching.](https://static.igem.wiki/teams/5760/results/thankyou1.webp)

## Major References

- Bao, L., Menon, P. N. K., Liljeruhm, J., & Forster, A. C. (2020). *Overcoming chromoprotein limitations by engineering a red fluorescent protein.* Analytical Biochemistry, 611, 113936. https://doi.org/10.1016/j.ab.2020.113936
- Chui, C. S. L., Cowling, B. J., Lim, W. W., Hui, C. K. M., Chan, E. W., Wong, I. C. K., & Wu, P. (2020). *Patterns of inpatient antibiotic use among public hospitals in Hong Kong from 2000 to 2015.* Drug Safety, 43(6), 595–606. https://doi.org/10.1007/s40264-020-00920-9
- Chung, S. S., Zheng, J. S., Burket, S. R., & Brooks, B. W. (2018). *Select antibiotics in leachate from closed and active landfills exceed thresholds for antibiotic resistance development.* Environment International, 115, 89–96. https://doi.org/10.1016/j.envint.2018.03.014
- Economist Impact. (2024, September). *Sea sickness: The pharmaceutical pollution problem.* Back to Blue Initiative. https://backtoblueinitiative.com/sea-sickness-the-pharmaceutical-pollution-problem/
- Ivantsova, N. A., Kuzin, E. N., Kuznetsov, V. V., Filatova, E. A., Pirogov, A. V., Timchenko, Y. V., Poineau, F., German, K. E., Averina, Y. M., & Kolesnikov, A. V. (2025). *Advanced electrochemical treatment of tetracycline‑contaminated water using Pt/Ti electrodes.* RSC Advances, 15(27), 21785–21793. https://doi.org/10.1039/d5ra02632f
- Leung, H. W., Jin, L., Wei, S., Tsui, M. M., Zhou, B., Jiao, L., Cheung, P. C., Chun, Y. K., Murphy, M. B., & Lam, P. K. (2013). *Pharmaceuticals in tap water: Human health risk assessment and proposed monitoring framework in China.* Environmental Health Perspectives, 121(7), 839–846. https://doi.org/10.1289/ehp.1206244
- Massana Roquero, D., & Katz, E. (2022). *“Smart” alginate hydrogels in biosensing, bioactuation and biocomputing: State‑of‑the‑art and perspectives.* Sensors and Actuators Reports, 4, 100095. https://doi.org/10.1016/j.snr.2022.100095
- Minh, T. B., Leung, H. W., Loi, I. H., Chan, W. H., So, M. K., Mao, J. Q., Choi, D., Lam, J. C., Zheng, G., Martin, M., Lee, J. H., Lam, P. K., & Richardson, B. J. (2009). *Antibiotics in the Hong Kong metropolitan area: Ubiquitous distribution and fate in Victoria Harbour.* Marine Pollution Bulletin, 58(7), 1052–1062. https://doi.org/10.1016/j.marpolbul.2009.02.004
- Wilkinson, J. L., Boxall, A. B. A., Kolpin, D. W., Leung, K. M. Y., Lai, R. W. S., Galbán‑Malagón, C., Adell, A. D., Mondon, J., Metian, M., Marchant, R. A., … Teta, C. (2022). *Pharmaceutical pollution of the world’s rivers.* Proceedings of the National Academy of Sciences of the United States of America, 119(8), e2113947119. https://doi.org/10.1073/pnas.2113947119
- Yang, W., Moore, I. F., Koteva, K. P., Bareich, D. C., Hughes, D. W., & Wright, G. D. (2004). *TetX is a flavin‑dependent monooxygenase conferring resistance to tetracycline antibiotics.* The Journal of Biological Chemistry, 279(50), 52346–52352. https://doi.org/10.1074/jbc.M409573200

![Scroll to Top Arrow](https://static.igem.wiki/teams/5760/button/arrow.webp)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

![iGEM Experiment Banner](https://static.igem.wiki/teams/5760/banner/2025-hongkong-jss-wiki-banner-zip-2-1.webp)

Our experiments are designed to be replicable, providing detailed protocols, materials, and conditions. Each section below outlines the purpose, materials, and step-by-step procedures to ensure clarity and reproducibility for future iGEM teams. Explore the wet lab and hardware experiments to understand our methodology, including detailed protocols for cloning, culturing, functional assays, and hardware testing.

## Wet Lab Experiments

---

### Part I: Chromoprotein Expression Plasmid Cloning

**Purpose:** To clone chromoprotein genes into pUC19 plasmids for expression in *E. coli*, enabling visual detection of successful cloning.

#### pUC19 Vector Preparation - Transformation

**Note:** This step transforms pUC19 into competent cells for plasmid amplification.

##### Materials:

* pUC19 positive control DNA (Invitrogen)
* One Shot™ TOP10 Chemically Competent *E. coli* (ThermoFisher, Cat# C404003) or DH5α Competent Cells (HKABE, CUHK)
* S.O.C Medium (ThermoFisher, Cat# 15544-034)
* LB agar plates with ampicillin (ThermoFisher, Cat# 22700-025; Gibco™, Cat# 11593027)

##### Procedures:

1. Mix 10 μL of pUC19 DNA with 1 mL of competent cells in a polypropylene tube stored at -80°C.
2. Incubate on ice for at least 10 minutes.
3. Heat shock at 42°C for 45 seconds.
4. Return to ice for 2 minutes.
5. Add 1 mL of pre-warmed S.O.C medium.
6. Incubate at 37°C for 10 minutes for recovery.
7. Spread 100-200 μL on an ampicillin LB agar plate.
8. Incubate at 37°C overnight.

#### Purification of pUC19 Plasmids

##### Materials:

* PureLink™ Quick Plasmid Miniprep Kit (ThermoFisher, Cat# K210010)

##### Procedures:

Follow the manufacturer’s manual. Use 50 μL of elution buffer for all preparations.

#### Restriction Digestion of pUC19

##### Materials:

* EcoRI-HF (NEB, Cat# R3101S)
* rCutSmart™ Buffer (NEB, Cat# B6004S)

##### Procedures:

| Component | 50 µL Reaction (µL) |
| --- | --- |
| DNA | 20 / 10 |
| rCutSmart™ Buffer | 5 |
| EcoRI-HF | 2 |
| Nuclease-free Water | 23 / 33 |

1. Incubate at 37°C for 30 minutes to 2 hours.
2. Heat inactivate at 65°C for 20 minutes.

#### Gel Purification of Digested pUC19

##### Materials:

* PureLink™ Quick Gel Extraction Kit (ThermoFisher, Cat# K210012)

##### Procedures:

Follow the manufacturer’s manual. Use 50 μL of elution buffer for all preparations.

#### HiFi Assembly

##### Materials:

* NEBuilder® HiFi DNA Assembly Master Mix (NEB, Cat# E2621S)

##### Procedures:

| Component | Chromoproteins (μL) | Negative Control (μL) | Positive Control (μL) |
| --- | --- | --- | --- |
| Vector | 2 | 10 | 10 |
| Insert | 6 | - | - |
| HiFi Master Mix | 10 | 10 | 10 |
| Nuclease-free Water | 2 | - | - |
| Total Volume | 20 | 20 | 20 |

Incubate at 50°C for 2 hours.

#### Colony PCR

##### Materials:

* M13 forward and reverse primers (100 μM, Tech Dragon)
* OneTaq® 2X Master Mix with Standard Buffer (NEB, Cat# M0482S)

##### Primers:

| Primers | Sequence |
| --- | --- |
| M13 forward | TGTAAAACGACGGCCAGT |
| M13 reverse | CAGGAAACAGCTATGACCATG |

##### Procedures:

Prepare master mix as follows:

| Components | X1 (µL) |
| --- | --- |
| Forward M13 | 0.5 |
| Reverse M13 | 0.5 |
| Template | Colonies |
| 2X OneTaq | 12.5 |
| H2O | 11.5 |
| Total | 25 |

Run PCR for 30 cycles with:

| Stage | Temperature (°C) | Time (s) |
| --- | --- | --- |
| Initial Denaturation | 94 | 30 |
| Denaturation | 94 | 20 |
| Annealing | 55 | 40 |
| Extension | 68 | 60 |
| Final Extension | 68 | 300 |

#### Gel Electrophoresis

##### Materials:

* The MiniOne Electrophoresis System (MiniOne Systems, Cat# M1000)
* Gel Loading Dye Purple (6X) (NEB, Cat# B7024S)
* UltraPure™ Agarose (Invitrogen, Cat# 16500500)
* TAE Buffer (Tris-acetate-EDTA, 50X) (ThermoFisher, Cat# B49)
* Quick-Load® Purple 1kb Plus DNA Ladder (NEB, Cat# N0550S)
* GelGreen™ DNA Stain 50 µL (MiniOne Systems, Cat# M3113)

##### Procedures:

1. Add 5 μL of loading dye to each DNA tube.
2. Add 25 μL distilled water and 5 μL loading dye to a new tube (negative control).
3. Set up gel tank:

* Prepare 1X TAE buffer for gel and tank.
* Prepare 1% agarose gel with 1X TAE buffer.
* Microwave gel for 1 minute to dissolve agarose.
* Cool flask with running water.
* Add 2 μL GelGreen™ DNA Stain per 20 mL gel.
* Transfer formed gel to tank and cover with buffer.

4. Add DNA samples and ladder to wells.

### Part II: *E. coli* Culturing

**Purpose:** To prepare and culture *E. coli* strains for downstream experiments.

#### *E. coli* Growth Media

##### Materials:

* LB Broth Base (Lennox) (Invitrogen, Cat# 12780052)
* LB Agar (Lennox L Agar), powder (Invitrogen, Cat# 22700025)
* Ampicillin, sodium salt, irradiated (Gibco™, Cat# 11593027)
* Kanamycin sulphate (Gibco™, Cat# 11815024)

##### Procedures:

1. Prepare media per manufacturer’s instructions using distilled water.
2. Sterilize using a pressure cooker (no autoclave available).
3. Add ampicillin or kanamycin after cooling.
4. Incubate cultures at 37°C, 180–200 rpm, unless otherwise noted.

### Part III: Functional Assay

**Purpose:** To test the functionality of biosensors and degradation systems for detecting and processing specific compounds.

#### Tetracycline Detection Assay

##### Materials:

* Tetracycline hydrochloride, 96% (ThermoScientific, Cat# B21408.14)
* Tetracycline stock solutions at 10 mg/mL, 1 mg/mL, 100 μg/mL

##### Procedures:

1. Grow starter culture in 10 mL LB/Amp broth for ≥2 hours.
2. Transfer 100 μL to 10 mL fresh LB/Amp broth, incubate ≥4 hours.
3. Add tetracycline stock to desired concentration (e.g., 10 μL of 100 μg/mL for 100 ng/mL).
4. Incubate ≥12 hours.
5. Observe color change. Centrifuge at >8000 g for 1 minute if needed.

#### Tetracycline Bead Detection Assay

##### Materials:

* Tetracycline hydrochloride, 96% (ThermoScientific, Cat# B21408.14)
* Tetracycline stock solutions at 10 mg/mL, 1 mg/mL, 100 μg/mL
* 2% Sodium alginate solution
* 2% Calcium chloride solution

##### Procedures:

1. Grow a starter culture of the cells in 10 mL fresh LB/Amp broth for at least 2 hours at 37°C, 180-200 rpm.
2. Transfer 100 μL of the starter culture to fresh 10 mL LB/Amp broth and incubate for at least 4 hours at 37°C, 180-200 rpm.
3. Mix the resulting culture with sodium alginate solution in a 1:1 volume ratio.
4. Use a dropper to add the mixture to calcium chloride solution drop by drop to form alginate beads containing the cells.
5. Prepare 10 mL LB/Amp culture medium with the desired concentration of tetracycline (e.g., 100 ng/mL).
6. Add the beads to the culture medium containing tetracycline.
7. Incubate for at least 12 hours at <30°C, 180-200 rpm.
8. Observe the color change of the beads against a white background. For clearer observation, replace the culture medium with ddH₂O.

#### Salicylate Detection Assay

##### Materials:

* Sodium salicylate, 99% (ThermoScientific, Cat# A17056.30)
* Salicylate stock solution at 100 mM

##### Procedures:

1. Grow starter culture in 10 mL LB/Amp broth for ≥2 hours.
2. Transfer 100 μL to 10 mL fresh LB/Amp broth, incubate ≥4 hours.
3. Add salicylate stock to desired concentration (e.g., 100 μL of 100 mM for 1 mM).
4. Incubate ≥12 hours.
5. Observe color change. Centrifuge at >8000 g for 1 minute if needed.

#### Tetracycline Degradation Assay

##### Materials:

* Tetracycline hydrochloride, 96% (ThermoScientific, Cat# B21408.14)
* Tetracycline stock solutions at 10 mg/mL, 1 mg/mL, 100 μg/mL

##### Procedures:

1. Grow starter culture in 10 mL LB/Amp broth for ≥2 hours.
2. Transfer 100 μL to 10 mL fresh LB/Amp broth, incubate ≥4 hours.
3. Blank spectrophotometer with 3 mL LB/Amp at 360 nm.
4. Add tetracycline stock to desired concentration (e.g., 50 μL of 1 mg/mL for 5 μg/mL).
5. Mix and transfer 1.5 mL to two 1.5 mL tubes.
6. Spin at 16,000 g for 5 minutes.
7. Take 3 mL supernatant into a cuvette, avoiding pellet.
8. Measure absorbance at 360 nm.
9. Repeat the step 6-8 at designated time points.

#### Tetracycline Standard Curve

##### Materials:

* Tetracycline hydrochloride, 96% (ThermoScientific, Cat# B21408.14)
* Tetracycline stock solutions at 10 mg/mL, 1 mg/mL, 100 μg/mL

##### Procedures:

1. Prepare tetracycline standards at 1, 2, 4, 6, 8, 10 μg/mL in LB/Amp.
2. Blank spectrophotometer with 3 mL LB/Amp at 360 nm.
3. Measure absorbance at 360 nm for each standard.
4. Record absorbances and use Gemini AI for calibration equation and R² value.

#### SDS-PAGE of Protein Lysates

##### Materials:

* 10X Bolt™ Sample Reducing Agent (Invitrogen™, Cat# B0009)
* 4X Bolt™ LDS Sample Buffer (Invitrogen™, Cat# B0007)
* 20X Bolt™ MES SDS Running Buffer (Invitrogen™, Cat# B0002)
* Bolt™ Bis-Tris Plus Mini Protein Gels, 4-12%, 1.0 mm (Invitrogen™, Cat# NW04120BOX)
* SeeBlue™ Plus2 Pre-stained Protein Standard (Invitrogen™, Cat# LC5925)
* SimplyBlue™ SafeStain (Invitrogen™, Cat# LC6060)
* Mini Gel Tank (Invitrogen™, Cat# A25977)

##### Procedures:

1. Harvest 500 μL overnight culture, spin at 12,000 g for 1 minute.
2. Discard supernatant, resuspend pellet in 26 μL ddH2O.
3. Mix 400 μL reducing agent with 1 mL LDS sample buffer.
4. Add 14 μL of the above buffer to sample, mix gently.
5. Heat at 95°C for 10 minutes.
6. Add 360 μL ddH2O, heat at 95°C for 10 minutes.
7. Place on ice for 5 minutes.
8. Prepare 1X MES SDS running buffer.
9. Set up gel system and pre-cast gel per manual.
10. Load 8 μL protein reference and 10 μL samples into wells.
11. Run gel at 150V for ~20 minutes.
12. Stain with SimplyBlue SafeStain, shake at 50 rpm for 20 minutes.
13. Destain in ddH2O until bands are visible, changing water if needed.

## Hardware Experiments

---

### Testing of Forming Alginate Beads

**Purpose:** To optimize conditions for forming alginate beads to encapsulate bacteria for biosensing applications.

#### Materials:

* Lead (II) Nitrate
* LB Broth
* Sodium Alginate
* Calcium Chloride

#### Procedures:

1. Prepare various concentrations of sodium alginate and calcium chloride solutions.
2. Mix 10 mL LB solution with sodium alginate solution in a 1:1 ratio.
3. Add LB–alginate solution to calcium chloride solution to form beads.
4. Test the following combinations:

| Conc. of NaAlg(aq) | Conc. of CaCl(aq) |
| --- | --- |
| 1:30 | 1:50 |
| 1:30 | 1:10 |
| 1:60 | 1:50 |
| 1:60 | 1:10 |

5. Examine formed alginate beads.

### Testing of Alginate Beads with Lead-Sensitive Bacteria

**Purpose:** To evaluate the functionality of lead-sensitive bacteria encapsulated in alginate beads for lead detection.

#### Materials:

* Lead (II) Nitrate
* LB Broth
* Ampicillin
* PbrR-pPbr lead biosensor (BBa\_K5152004)
* Sodium Alginate
* Calcium Chloride

#### Procedures:

1. Grow PbrR-pPbr lead biosensor in 10 mL LB broth with ampicillin for 18 hours.
2. Mix 10 mL LB solution with a 1:60 sodium alginate solution.
3. Add bacterial–alginate solution to a 1:10 calcium chloride solution to form beads.
4. Extract formed alginate beads.
5. Add beads to 10 mL LB broth with ampicillin containing 0 mM, 0.01 mM, 0.1 mM, 1 mM, 10 mM Lead (II) Nitrate.
6. Incubate at 37°C in a shaking incubator for 24 hours.
7. Extract beads and observe color change.

![Scroll to Top Arrow](https://static.igem.wiki/teams/5760/button/arrow.webp)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their
significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5760/banner/2025-hongkong-jss-wiki-banner-1.webp)

##### Contents

* [Cloning of Chromoprotein Expression Constructs](#chromoprotein-construction)
* [Comparing the Expression of Chromoproteins](#comparative-analysis)
* [Tetracycline Biosenseor Functional Assays](#biosensor-development)
* [Tetracucline Degradation Functional Assays](#degradation-studies)
* [Summary of Key Findings](#summary)

## Cloning of Chromoprotein Expression Constructs

### pUC19 Backbone Preparation

Our experimental workflow commenced with the construction of chromoprotein-expressing *E. coli*
strains. Initially, pUC19 plasmids were transformed into TOP10 *E. coli* cells via heat shock
transformation, followed by selection on LB/ampicillin plates. A single transformant colony was cultured
overnight and subjected to miniprep isolation to obtain sufficient pUC19 plasmid DNA.

The purified pUC19 was subsequently linearized through EcoRI restriction digestion. The initial digestion
reaction was performed according to the conditions specified below, with a 30-minute incubation period:

| Component | 50 μL Reaction Volume (μL) |
| --- | --- |
| DNA | 20 |
| 10X CutSmart Buffer | 5 |
| EcoRI-HF | 2 |
| Nuclease-free Water | 23 |

However, preliminary transformation results revealed numerous colonies lacking chromoprotein expression,
suggesting incomplete vector digestion using the above protocol. To achieve complete linearization, we
optimized the digestion conditions by reducing the DNA input and extending the incubation time:

| Component | 50 μL Reaction Volume (μL) |
| --- | --- |
| DNA | 10 |
| 10X CutSmart Buffer | 5 |
| EcoRI-HF | 2 |
| Nuclease-free Water | 33 |

The incubation period was extended to 2 hours to ensure complete digestion.

### Insert Preparation

Assembly of chromoprotein inserts into the linearized vector was accomplished using the HiFi DNA Assembly
method, facilitated by the generous sponsorship of the HiFi Assembly Kit from New England Biolabs (NEB).

Chromoprotein gene sequences were synthesized as gBlocks by Integrated DNA Technologies (IDT). The
following expression constructs were ordered:

* Constitutive mRFP1e expression
* Constitutive aeBlue expression
* Constitutive SYFP2 expression
* Constitutive mRFP1e + aeBlue co-expression
* Constitutive SYFP2 + mRFP1e co-expression
* Constitutive SYFP2 + aeBlue co-expression

Each lyophilized gBlock was resuspended to a final concentration of 100 ng/μL.

### HiFi Assembly of Chromoprotein Expression Constructs

Due to the unavailability of spectrophotometric quantification, pUC19 vector concentration was estimated
by comparison to a DNA ladder during agarose gel electrophoresis. Assembly reaction ratio was
calculated with AI assistance, determining that approximately 5.5-6 μL of insert was required
per 2 μL of vector. For consistency, all insert volumes were standardized to 6 μL.

The assembly reaction composition was as follows:

| Component | Chromoproteins Inserts(μL) | Negative Control (μL) | Positive Control (μL) |
| --- | --- | --- | --- |
| Vector | 2 | 10 | 10 |
| Insert | 6 | - | - |
| HiFi Master Mix | 10 | 10 | 10 |
| Nuclease-free Water | 2 | - | - |
| Total Volume | 20 | 20 | 20 |

The assembly mixtures were incubated at 55°C for 2 hours to facilitate DNA joining. Following assembly,
the reaction products were transformed into chemically competent TOP10 *E. coli* cells using the standard
heat shock transformation protocol. Transformants were selected on LB agar plates supplemented with
ampicillin to maintain plasmid selection pressure.

### Successful Construction of Chromoprotein-Expressing *E. coli* Strains

After 24 hours of incubation at 37°C, several pigmented colonies were observed on the transformation
plates, confirming the successful construction of chromoprotein-expressing bacterial strains. The
appearance of colored colonies provided direct visual evidence of functional chromoprotein expression and
proper assembly of the expression constructs.

However, transformants expressing SYFP2 required visualization under blue light excitation to observe the
characteristic yellow fluorescence, as reported in previous studies. Since our objective was to identify
chromoproteins that produce readily distinguishable colors under ambient lighting conditions for naked-eye
detection, SYFP2 did not meet our selection criteria. Consequently, further characterization of
SYFP2-expressing strains was discontinued, and efforts were focused on the more visually apparent
chromoproteins.

The transformation results are presented in the figures below:

![Fig 1. Chromoprotein-expressing bacterial colonies on selective media following transformation. Pigmented colonies displaying distinct coloration patterns are visible on ampicillin-containing agar plates, demonstrating successful expression of the engineered chromoprotein constructs in <i>E. coli</i> TOP10 cells.](https://static.igem.wiki/teams/5760/results/1-1.webp)

Fig 1. Chromoprotein-expressing bacterial colonies on selective media
following transformation. Pigmented colonies displaying distinct coloration patterns are visible on
ampicillin-containing agar plates, demonstrating successful expression of the engineered chromoprotein
constructs in *E. coli* TOP10 cells.

![Fig 2. SYFP2-expressing <i>E. coli</i> colony visualized under blue light excitation. A single transformant colony exhibits bright yellow fluorescence, confirming successful expression of the SYFP2 fluorescent protein construct.](https://static.igem.wiki/teams/5760/results/2-1.webp)

Fig 2. SYFP2-expressing *E. coli* colony visualized under blue light
excitation. A single transformant colony exhibits bright yellow fluorescence, confirming successful
expression of the SYFP2 fluorescent protein construct.

## Comparing the expression of dTomato and mRFP1e

We performed a comparative study of dTomato and mRFP1e expression to determine which chromoprotein
provides better visual detection as a reporter protein. Both chromoproteins were expressed using identical
genetic regulatory components, including the same promoter, RBS, and terminator sequences. This
standardized approach ensured that any differences in expression or coloration could be directly
attributed to the specific characteristics of each chromoprotein rather than variations in gene
regulation.

Both bacterial cultures were grown under the same conditions at 37°C for 14 hours with shaking at 180 rpm
to maintain consistent growth and protein expression environments.

![Fig 3. Comparative expression analysis of mRFP1e and dTomato chromoproteins. mRFP1e displays deeper red coloration than dTomato in both agar plate cultures and liquid medium following 14 hours of incubation.](https://static.igem.wiki/teams/5760/results/3-1.webp)

Fig 3. Comparative expression analysis of mRFP1e and dTomato chromoproteins.
mRFP1e displays deeper red coloration than dTomato in both agar plate cultures and liquid medium
following 14 hours of incubation.

Based on the results shown in Fig. 3, mRFP1e demonstrated better color intensity compared to dTomato
under our experimental conditions. The deeper red pigmentation produced by mRFP1e makes it a more suitable
candidate for biosensor applications, as stronger visual signals provide better detection sensitivity and
easier identification without specialized equipment.

### An Interesting Observation from the Coexpression Strain

We noticed something interesting during our experiments that caught our attention. We consistently
observed that the mRFP1e+aeBlue coexpression strain produced deeper coloration than the strain expressing
mRFP1e alone. This observation sparked our curiosity, so we decided to compare their expression levels
directly.

![Fig 4. Comparative expression analysis of mRFP1e+aeBlue coexpression and mRFP1e expression strains. The coexpression strain displays deeper red coloration than the mRFP1e-only expression strain in both agar plate cultures and liquid medium following 12 hours of incubation.](https://static.igem.wiki/teams/5760/results/4-1.webp)

Fig 4. Comparative expression analysis of mRFP1e+aeBlue coexpression and
mRFP1e expression strains. The coexpression strain displays deeper red coloration than the mRFP1e-only
expression strain in both agar plate cultures and liquid medium following 12 hours of incubation.

The figure above shows cells after 12 hours of expression at 37°C, 180 rpm. We can clearly see that the
color intensity of the coexpression strain is noticeably higher than the mRFP1e-only strain in both agar
plate cultures and liquid medium. This was quite an interesting finding, though we chose not to pursue its
application further. After consulting with our teacher, we learned that expressing two proteins
simultaneously may overload the bacterial cells, which could actually decrease the sensitivity of our
biosensor system if implemented in the final design.

### SDS-PAGE Analysis of mRFP1e and mRFP1e+aeBlue Coexpression

Out of curiosity, we collected cell lysates for further analysis. The gel diagram is shown below.

![Fig 5. SDS-PAGE analysis of chromoprotein expression. Cell lysates from mRFP1e and mRFP1e+aeBlue expressing cultures were separated by SDS-PAGE. An additional band of unknown identity is visible in the coexpression sample compared to controls.](https://static.igem.wiki/teams/5760/results/5-1.webp)

Fig 5. SDS-PAGE analysis of chromoprotein expression. Cell lysates from mRFP1e
and mRFP1e+aeBlue expressing cultures were separated by SDS-PAGE. An additional band of unknown identity
is visible in the coexpression sample compared to controls.

The results showed that an unexpected band appears in the lysate of coexpression cells, which is
approximately 30 kDa in size and does not match either mRFP1e or aeBlue. We hypothesize that this is due
to our use of overlapping start and stop codons at the junction to coexpress the proteins. The stop codon
may not function efficiently, leading to poor translation termination and resulting in a larger protein
product. Since this was not the focus of our project, we did not conduct any further investigation.

## Detecting the best concentration of induction of Tetracycline biosensor

To find out the best concentration of tetracycline (Tc) to induce our biosensor, we performed a simple
experiment to see the growth of *E. coli* in a series of dilutions of Tc.

Concentrations ranging from ~38 ng/mL to 600 ng/mL Tc with 2-fold increments were cultured with *E.
coli* along with a control to observe their growth. After 14 hours of incubation at 37°C, as shown in
the figure below, cultures with ~300 ng/mL and ~600 ng/mL Tc showed significant growth inhibition.

![Fig 6. Growth inhibition of <i>E. coli</i> at different tetracycline concentrations. <i>E. coli</i> cultures were grown in the presence of tetracycline at concentrations ranging from ~38 ng/mL to 600 ng/mL with 2-fold increments, along with a control (no tetracycline). Significant growth inhibition was observed at ~300 ng/mL and ~600 ng/mL tetracycline concentrations, indicating the cytotoxic effects of higher tetracycline concentrations on bacterial growth.](https://static.igem.wiki/teams/5760/results/6-1.webp)

Fig 6. Growth inhibition of *E. coli* at different tetracycline
concentrations. *E. coli* cultures were grown in the presence of tetracycline at concentrations
ranging from ~38 ng/mL to 600 ng/mL with 2-fold increments, along with a control (no tetracycline).
Significant growth inhibition was observed at ~300 ng/mL and ~600 ng/mL tetracycline concentrations,
indicating the cytotoxic effects of higher tetracycline concentrations on bacterial growth.

Based on the above result, we decided to use 200 ng/mL as the maximum detection concentration.

### Functional Study on Biosensors

#### Functional Assays on Salicylate Detection

**Design 1 & 2: [BBa\_25F9SR86](https://registry.igem.org/parts/bba-25f9sr86)
and [BBa\_25WAI7EY](https://registry.igem.org/parts/bba-25WAI7EY)**

We designed and ordered two salicylate biosensor constructs, *NahR/pSal* [BBa\_25F9SR86](https://registry.igem.org/parts/bba-25f9sr86) and [BBa\_25WAI7EY](https://registry.igem.org/parts/bba-25wai7ey), in pIDT-Amp vector
from IDT and pTwist-Amp from Twist Bioscience respectively. The plasmids were then transformed into both
TOP10 and BL21 cells for functional testing.

Individual colonies were inoculated into 10 mL LB/Amp starter cultures and grown for 4 hours at 37°C with
180 rpm shaking. We then transferred 100 μL of starter culture into 10 mL fresh LB/Amp medium with
salicylate concentrations from 0.1 mM to 2 mM and continued growing at 37°C with 180 rpm shaking. We
monitored color changes at 6, 16, 24, and up to 36 hours after induction. At each time point, we collected
1 mL of culture and centrifuged at 8000 g for 1 minute to form cell pellets for better color observation.

Unfortunately, no color changes occurred in any biosensor setup. We tried troubleshooting with different
inducer concentrations, longer incubation times, lower temperatures, and different cell strains like BL21.
Despite these attempts, we saw no biosensor response, suggesting the problem was with our construct design
rather than the experimental conditions.

#### Functional Assays on Tetracycline Detection

**Design 1: [BBa\_254KMUU6](https://registry.igem.org/parts/bba-254KMUU6)**

This biosensor design, similar to the salicylate biosensor above, showed no response to tetracycline (Tc)
under any testing conditions, so we moved on to a new design.

**Design 2: [BBa\_252ZMUOK](https://registry.igem.org/parts/bba-252ZMUOK)**

The tetracycline biosensor successfully detected tetracycline through a visible blue color change due to
*amilCP* reporter gene expression.

In this *TetR/pTet* biosensor design, after adding 100 ng/mL tetracycline and incubating for 18
hours at 25 °C with 180 rpm shaking, a significant blue color was observed in the biosensor cells. The
color became deeper with 200 ng/mL added, indicating a concentration-dependent effect. The controls,
including no Tc (0 ng/mL Tc) and pUC19 (added with 100 ng/mL Tc), showed no visible coloration to the
naked eye, as shown in the figure below.

However, with 10 ng/mL Tc added, the biosensor didn't show a visible color change indicating no response,
meaning the sensitivity of the biosensor is unable to detect this concentration range.

![Fig 7. Tetracycline detection by <i>TetR/pTet</i> biosensor (BBa_252ZMUOK). Cell pellets showing concentration-dependent blue color development in response to tetracycline. However, the biosensor shows sensitivity limits with no detectable response at 10 ng/mL concentration.](https://static.igem.wiki/teams/5760/results/7-1.webp)

Fig 7. Tetracycline detection by *TetR/pTet* biosensor ([BBa\_252ZMUOK](https://registry.igem.org/parts/bba-252ZMUOK)). Cell pellets
showing concentration-dependent blue color development in response to tetracycline. However, the
biosensor shows sensitivity limits with no detectable response at 10 ng/mL concentration.

To further validate the results and better observe the response of the biosensor, we performed another
set of experiments using smaller concentration intervals (0, 10, 50, 100, 150, and 200 ng/mL) of Tc to
induce the biosensors. The results (Fig. 7) showed that when compared with the empty vector pUC19 control
and no Tc control, the cell pellets treated with Tc starting from 50 ng/mL concentration showed a
noticeable blue color from amilCP chromoprotein that was visibly different from the controls by naked eye
observation. The color intensity increased with increasing Tc concentration.

![Fig 8. Another set of replicates using smaller intervals of tetracycline concentrations to assess the response of our biosensor.](https://static.igem.wiki/teams/5760/results/8-1.webp)

Fig 8. Another set of replicates using smaller intervals of tetracycline
concentrations to assess the response of our biosensor.

However, from this set of results, we observed that the color intensity of the pellets did not resemble
that of the first set. This was because we used an overnight culture diluted for the experiment due to
time constraints, instead of using a freshly prepared culture. This may have caused an unhealthy status of
our biosensors, leading to these results. This made us aware that we should better maintain the
biosensor's growth conditions.

Despite these limitations, distinct color variations were still evident between the tetracycline-exposed
biosensors and control samples. These findings provide additional confirmation of our biosensor's
operational capacity and support the validity of our design approach.

#### SDS-PAGE validation of the expression

We also performed SDS-PAGE to further validate the expression of the biosensor construct. The whole
protein lysate of the biosensor culture was run on a 4-12% Bis-Tris Plus Gel with MES SDS running buffer
at 150V. The gel was stained with Coomassie Blue. The protein ladder used was SeeBlue Plus2 Pre-Stained
Standard.

![Fig 9. SDS-PAGE analysis confirming TetR protein expression in the biosensor construct. A distinct band at ~23 kDa was observed in the biosensor lane, corresponding to the expected molecular weight of TetR protein.](https://static.igem.wiki/teams/5760/results/9-2.webp)

Fig 9. SDS-PAGE analysis confirming TetR protein expression in the biosensor
construct. A distinct band at ~23 kDa was observed in the biosensor lane, corresponding to the expected
molecular weight of TetR protein.

From the gel photo above, when compared with the pUC19 control, a distinct band was observed at ~23 kDa
in the lane containing our biosensor, which matches the size of the TetR protein expressed by the
biosensor and is absent in the control. This result strongly validates the successful creation of our
biosensor.

#### Bead-Based Biosensor Application

To validate the plausibility of our concept of using alginate beads to immobilize biosensors, we produced
beads containing our biosensor and incubated them in LB/Amp culture with or without tetracycline (100
ng/mL or 200 ng/mL). The results are shown below.

![Fig 10. Alginate bead-based tetracycline biosensor response. Beads containing biosensor cells or pUC19 control were incubated with varying tetracycline concentrations (0-200 ng/mL) for 16 hours. Visible color changes indicate successful tetracycline detection in the bead format.](https://static.igem.wiki/teams/5760/engineering/beads2.avif)

Fig 10. Alginate bead-based tetracycline biosensor response. Beads containing
biosensor cells or pUC19 control were incubated with varying tetracycline concentrations (0-200 ng/mL)
for 16 hours. Visible color changes indicate successful tetracycline detection in the bead format.

The figure demonstrates visually detectable color alterations in beads treated with 100 ng/mL and 200
ng/mL tetracycline-containing medium compared to both pUC19 empty vector controls and tetracycline-free
controls following 16-hour incubation. These findings confirm the feasibility of our bead-based biosensor
approach.

## Functional Study on Degradation Strains

### Functional Assays on Salicylate Degradation

For salicylate degradation testing, since we lacked UV-spectrophotometers to measure salicylate or product
concentrations, we attempted an alternative approach using FeCl₃·6H₂O, which reacts with salicylate to
form purple compounds measurable at 530 nm. However, we could not construct a reliable standard curve,
likely due to our expired FeCl₃·6H₂O stock forming Fe³⁺ hydroxide over time. Due to time constraints, we
abandoned this quantification approach.

Nevertheless, we ran SDS-PAGE to demonstrate the successful expression of NahG and XylE. The gel diagram
is shown below:

![Fig 11. SDS-PAGE showing the protein expression in our constructs. In the lane of NahG/XylE expression cells' proteins, two thickened bands can be observed at the positions matching NahG (~49.3 kDa) and XylE (~35.1 kDa).](https://static.igem.wiki/teams/5760/results/11-1.webp)

Fig 11. SDS-PAGE showing the protein expression in our constructs. In the lane
of NahG/XylE expression cells' proteins, two thickened bands can be observed at the positions matching
NahG (~49.3 kDa) and XylE (~35.1 kDa).

From the gel results below, we can see that in the lane containing lysates from NahG/XylE-expressing
cells, two distinct bands appear at positions corresponding to NahG (~49.3 kDa) and XylE (~35.1 kDa),
indicating successful expression of both proteins. Moreover, the XylE band shows noticeably higher
intensity than the NahG band, confirming our design concept that XylE is expressed at a higher level
than NahG. This likely results in greater XylE activity within the cells, helping to prevent catechol
accumulation during salicylate degradation.

### Functional Assays on Tetracycline Degradation

A standard curve using absorbance at 360 nm was constructed to measure tetracycline (Tc) concentration in
culture medium by spectrophotometer. LB/Amp solution without Tc was used as blank.

Standard LB/Amp solutions with Tc concentrations of 1, 2, 4, 6, 8, and 10 μg/mL were prepared. The
absorbance results showed a linear relationship with the equation y = 0.0294x - 0.0041 and R² = 0.999,
indicating high reliability. This validates the measurement method for monitoring Tc concentration changes
in LB culture medium.

| Tc Concentration (μg/mL) | Abs₃₆₀ |
| --- | --- |
| 0 | 0 |
| 1 | 0.025 |
| 2 | 0.057 |
| 4 | 0.115 |
| 6 | 0.177 |
| 8 | 0.229 |
| 10 | 0.290 |

![Fig 12. Standard curve for tetracycline concentration measurement. Linear relationship between tetracycline concentration (μg/mL) and absorbance at 360 nm in LB/Amp medium.](https://static.igem.wiki/teams/5760/results/12.webp)

Fig 12. Standard curve for tetracycline concentration measurement. Linear
relationship between tetracycline concentration (μg/mL) and absorbance at 360 nm in LB/Amp medium.

The linear equation was then used to calculate Tc concentration in the culture medium for subsequent
degradation experiments.

### Tetracycline Degradation Assay

Cells expressing TetX were grown in LB/Amp medium for 2 hours. We transferred 100 μL starter culture to
10 mL degradation culture and incubated for 4-6 hours until log phase. Tetracycline was added to final
concentrations of ~5 μg/mL and ~10 μg/mL. We immediately measured baseline absorbance (time = 0) by
centrifuging cells at 12,000 g for 5 minutes and measuring supernatant absorbance at 360 nm. Subsequent
measurements were taken at 2 and 4 hours post-addition.

The results are shown in the tables below, with absorbance values converted into concentration using the
standard curve equation.

**Table 1.** Degradation result of tetracycline by TetX expressing cells (5 μg/mL
initial concentration)

| Time (hr) | LB + Tet (CTRL) | E. coli + Tet (CTRL) | TetX + Tet |
| --- | --- | --- | --- |
| 0 | 6.33 | 7.62 | 6.4 |
| 2 | 5.89 | 7.55 | 2.86 |
| 4 | 6.13 | 7.72 | 2.93 |

**Table 2.** Degradation result of tetracycline by TetX expressing cells (10 μg/mL
initial concentration)

| Time (hr) | LB + Tet (CTRL) | E. coli + Tet (CTRL) | TetX + Tet |
| --- | --- | --- | --- |
| 0 | 10.62 | 11.6 | 10.17 |
| 2 | 10.85 | 11.91 | 5.68 |
| 4 | 10.45 | 11.98 | 5.79 |

![Fig 13. Time-course analysis of tetracycline degradation by TetX cells over 2 and 4 hours.](https://static.igem.wiki/teams/5760/results/13.webp)

Fig 13. Time-course analysis of tetracycline degradation by TetX cells over 2
and 4 hours.

The results demonstrated that TetX cells significantly decreased tetracycline concentration in the
culture medium, while control setups (pUC19 empty vector cells and LB/amp medium only) maintained their
tetracycline levels. This shows that TetX cells are effectively working as expected, with results further
supported by SDS-PAGE showing TetX enzyme expression.

We observed that after 4 hours, tetracycline concentration in TetX cultures did not further decrease. We
suspect this was due to prolonged incubation, causing cells to enter post-log phase and reduced
tetracycline substrate concentration, greatly reducing TetX enzyme activity after 2 hours. To test this
hypothesis, we repeated the experiment using a shorter time frame.

Using the same methods as the previous experiment, absorbance changes were measured every 30 minutes over
a 2-hour time frame to monitor tetracycline concentration changes in TetX cell cultures compared to
controls.

**Table 3.** Tetracycline degradation by TetX-expressing cells over a 2‑hour
period with measurements taken at 30‑minute intervals

| Time (hr) | LB only (Rep 1) (µg/mL) | LB only (Rep 2) (µg/mL) | pUC19 + Tet (Rep 1) (µg/mL) | pUC19 + Tet (Rep 2) (µg/mL) | TetX + Tet (Rep 1) (µg/mL) | TetX + Tet (Rep 2) (µg/mL) |
| --- | --- | --- | --- | --- | --- | --- |
| 0 | 6.74 | 6.74 | 6.4 | 7.01 | 6.02 | 5.62 |
| 0.5 | 5.17 | 6.36 | 6.36 | 7.42 | 4.97 | 4.49 |
| 1 | 5.31 | 6.4 | 6.64 | 7.15 | 4.19 | 3.74 |
| 1.5 | 5.14 | 6.23 | 6.53 | 7.11 | 3.78 | 3.44 |
| 2 | 5.28 | 6.33 | 6.43 | 7.21 | 3.44 | 2.93 |

![Fig 14. Tetracycline degradation assay over 2 hours. Three conditions were tested: LB only, pUC19 control + tetracycline, and TetX-expressing cells + tetracycline. Error bars represent SD of two biological replicates (n=2).](https://static.igem.wiki/teams/5760/engineering/degra2.avif)

Fig 14. Tetracycline degradation assay over 2 hours. Three conditions were
tested: LB only, pUC19 control + tetracycline, and TetX-expressing cells + tetracycline. Error bars
represent SD of two biological replicates (n=2).

As shown in the results, the tetracycline concentration in the culture medium of TetX cells continued to
decrease at every time point, indicating that TetX is actively degrading tetracycline within this
timeframe. This result further confirms the functionality of our tetracycline degradation cells and
enhances our understanding of their tetracycline degradation kinetics. In the graph, the error bars
represent the standard deviation of two replicates from individual cultures of TetX cells.

In the results above, we observed substantial variation in absorbance values,
including initial readings and replicates, despite maintaining careful and consistent technique.
We suspect that interference from cells and other components in the LB medium contributed to this
variability. Since the reported absorbance 360 nm measuremnt method for tetracycline detection was developed in pure water,
it may not fully apply to our conditions. The complex composition of the LB/Amp medium and the presence
of cells likely introduced instability in the measurements. Nonetheless, the pronounced decrease in
absorbance observed in TetX-expressing strains, compared to the relatively stable control, still provides
strong evidence that our degradation cells are functioning effectively.

### SDS-PAGE Confirmation of TetX Expression

SDS-PAGE analysis was performed following the protocols outlined in the experiment section. Protein
lysates were prepared from the TetX cultures used in the degradation experiments and analyzed alongside
lysates from *E. coli* control and pUC19 empty vector control. The expected molecular weight of TetX
protein is approximately 44 kDa.

The SDS-PAGE results clearly demonstrate a distinct band at ~44 kDa in the TetX sample that is not
present in either the *E. coli* or pUC19 control samples. This confirms successful expression of the
TetX enzyme in our degradation strain.

![Fig 15. SDS-PAGE analysis confirming TetX protein expression in the degradation strain.](https://static.igem.wiki/teams/5760/results/15.webp)

Fig 15. SDS-PAGE analysis confirming TetX protein expression in the
degradation strain.

## Summary of Key Findings

1. We successfully designed and created chromoprotein expression strains for mRFP1e, aeBlue, SYFP2, and a
   coexpression strain combining mRFP1e and aeBlue, demonstrating their distinct color properties.
2. Our preliminary studies comparing dTomato and mRFP1e chromoproteins revealed that mRFP1e exhibits
   superior characteristics for biosensor applications due to its faster maturation kinetics.
3. Additionally, when comparing mRFP1e alone versus the mRFP1e and aeBlue coexpression strain, we observed
   that the coexpression system consistently displayed accelerated maturation and enhanced color intensity,
   though the underlying mechanism remains unclear.
4. The tetracycline biosensor ([BBa\_25A81M7M](https://registry.igem.org/parts/bba-25A81M7M)) successfully detected tetracycline presence through visible color
   changes, with color intensity correlating to tetracycline concentration. This demonstrates the potential
   for real-world diagnostic applications of these engineered strains.
5. Our tetracycline biosensor cells were effectively immobilized using calcium-alginate encapsulation
   technology. Viability assays confirmed that the entrapped cells retained their metabolic activity and
   tetracycline-sensing capabilities, displaying distinct chromoprotein responses upon target detection.
   This demonstration validates the potential of alginate-based cell immobilization as a practical method
   for developing portable, contained biosensor platforms suitable for field applications.
6. Our tetracycline degradation remediator ([BBa\_25CS26NB](https://registry.igem.org/parts/bba-25cs26nb)) strains demonstrated their capability to reduce tetracycline concentrations in the extracellular environment, highlighting their potential as an effective biological approach for mitigating tetracycline pollution in real-world applications.
7. We successfully designed and expressed the NahG/XylE salicylate degradation construct ([BBa\_257IE4RA](https://registry.igem.org/parts/bba-257ie4ra)).
   Although we did not directly verify its degradation activity,
   SDS-PAGE analysis confirmed the expression of both NahG and XylE proteins. Moreover,
   our results demonstrated that XylE was expressed at a higher level than NahG, supporting
   our design concept that a higher XylE activity-to-NahG ratio can help prevent
   the accumulation of the toxic intermediate catechol during salicylate degradation.

These results informed our strategic planning sessions, where we identified key areas for improvement and
established a roadmap for project advancement. Our future efforts will prioritize enhancing biosensor
sensitivity and resolving the technical limitations we observed. A detailed account of our iterative
design process, including the obstacles faced and solutions implemented during biosensor development, can
be found on our Engineering page.

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