Integrating Human Practices into the Bye-o-Film project is the effort of having an iterative dialogue between stakeholders and the project team to discuss the wants and needs of our stakeholders and to weigh their desires and instructions. All with the goal of appropriately changing and redefining the Bye-o-Film project's scope and ambitions along the way.

Through our interactions under the guise of Human Practices, we explore how our project affects society and how it should be influenced by it. Our conversations with patients, scientific experts, and commercial companies have offered a continuous feedback loop that guided our work throughout the iGEM competition. Throughout our Wiki meaningful interactions can be found and at their respective sections. The impact of particular interactions is well described, but all these interactions are also combinedly described in our [Human Practices] section

Figure 2: Visual overview of the main goals regarding Integrated Human Practices within the Bye-O-Film project. Created using the resources from Slidesgo and Freepik.

Our project serves as a beacon of inspiration for the synthetic biology community. We demonstrate a holistic approach to Human Practices, extending beyond the lab. By actively engaging with stakeholders and documenting our experiences, we set a high standard for ethical and responsible synthetic biology projects. Other teams can draw from our methods, learning how to identify and establish meaningful connections with stakeholders, conduct insightful surveys and interviews, and navigate ethical complexities. Our dedication to improving patient outcomes resonates as a testament to the positive impact synthetic biology can have on society.

Our Human Practices implementation is characterized by careful consideration. We've provided clear context and rationale for engaging with stakeholders, bolstered by literature references and expert consultations. Emphasizing the importance of diverse perspectives, we've actively reached out through surveys and interviews to identify stakeholder characteristics and expectations related to the Bye-o-Film project. Our commitment to ongoing engagement beyond the competition showcases our dedication to aligning our project with societal needs, ensuring a strong foundation and goal alignment.

Our biosensor comprises three interchangeable modules: the sensor, reporter, and effector modules. To highlight the versatility of our design, we even proposed an alternative luciferase-based reporter module, providing future teams with a strong foundation for biosensor development. During our brainstorming process, we came across the cyclic-di-GMP-inducible promoter designed by the CUG China 2022 iGEM team, that we wished to incorporate into our modules, to be able to activate them when biofilms are being formed. Our vision was to use this promoter in our design and optimize it according to our needs, further adapting it for its possible future applications. Below we discuss the efforts and achievements we have made during working towards this goal.

The design improvements made to the original CUG-China promoter involve several modifications and optimization steps.

1. Codon Optimization and RFC [1000] Compatibility

The FleQ coding sequence underwent codon optimization to enhance its expression. During this process, three SapI restriction sites were removed, making the part compatible with RFC[1000] standards, ensuring its usability in synthetic biology assembly.

2. SapI Golden Gate Assembly Attempts

Initially, synthetic DNA fragments were ordered and designed with 30 bp overlaps for Gibson Assembly into the pSB1A3 vector. However, this approach faced challenges as the FleQ coding sequence consistently went missing in the screening results of colonies transformed with the Gibson Assembly reaction mix. To address this, SapI Golden Gate Assembly was attempted using codon-optimized fragments, but this also resulted in difficulties, with some colonies containing empty backbones and others only the promoter insert.

3. Investigation of Recombination Issues

A recurring problem appeared to be related to the repeating sequence of the PcI promoter, present in front of both FleQ and the PpeI promoter. This repetition caused recombination events, leading to the deletion of FleQ. It was hypothesized that the circularization of FleQ due to a mismatch in the sticky ends might be causing this issue.

4. Transition to BsaI Golden Gate Assembly

To overcome the recombination problem, a new design was introduced for Golden Gate Assembly with BsaI. BsaI cleavage resulted in 4-base sticky ends instead of 3-base ends with SapI, increasing the number of possible combinations. The high-strength BBa_J23100 promoter replaced the Pcl promoter in front of FleQ to eliminate the repeating sequence. The fragments required for assembly were successfully obtained by Q5 PCR, but challenges persisted, with colonies from the transformation showing no correct fragment sizes.

5. Further Efforts and Replacement of Terminator Sequences

To address recombination and achieve the desired construct, it was evident that one of the termination sequences needed replacement. Specific terminator sequences suitable for the biosensor module were proposed, acknowledging the need for a new terminator as FleQ also contained terminator sequences.

In summary, the improvements made to the original CUG-China promoter involved codon optimization, compatibility enhancements, transitions between assembly methods (Gibson to SapI Golden Gate to BsaI Golden Gate), and the replacement of terminator sequences to overcome recombination issues, ultimately aiming to create a more functional and reliable cyclic-di-GMP sensor module.

While our documentation showcased design enhancements, such as the removal of repetitive sequences and codon optimization, we must acknowledge that we did not present experimental data directly comparing the functionality of BBa_K4720021 to BBa_K4242013. We recognize the importance of future experiments to establish any enhancements in performance or reliability resulting from our improvements. Using BBa_K4242013 as a control in these experiments will be essential.

Our engineering process emphasized iterative improvements, optimizing promoter strengths, normalizing reporter expression, and preventing undesirable recombination events. This modularity allows for future adaptability, aligning with our commitment to a flexible and versatile biosensor. The information provided details our design and assembly processes for BBa_K4720021, including cloning strategies and primer sequences. Yet, it's crucial to acknowledge the absence of experimental data characterizing its functionality. To comprehensively characterize this improved part, experiments evaluating its performance, sensitivity, and specificity as a biosensor should be conducted. Sharing these experimental results on the iGEM Registry will be instrumental in facilitating broader use within the iGEM community. Our team succeeded in emphasizing iterative improvements, optimizing promoter strengths, normalizing reporter expression, and preventing undesirable recombination events. This modularity allowed future adaptability, aligning with our commitment to a flexible and versatile biosensor.

Despite encountering challenges, particularly related to FleQ functionality and cyclic-di-GMP response during testing, we maintained a systematic troubleshooting approach. Our future endeavors include exploring directions like incorporating our biosensor into the genome using CRISPR technology, reflecting our dedication to continual growth and refinement, ensuring our biosensor remains a valuable resource for the iGEM community. Below we detail our engineering process to showcase the efforts we made towards achieving this design goal.

Figure 5: Visual overview of the Key steps in our design process, generating knowledge for the community. Created using the resources from Slidesgo and Freepik.

In the initial stages of our design process, our focus centered on the meticulous crafting of various crucial constructs, each with its distinct role within our biosensor framework. These constructs encompassed reporter constructs, denoted as Construct 1, 1B, and 1C, sensor constructs under the banner of Construct 3, and effector constructs represented by Construct 5, as detailed on the [engineering] page. Each of these intricate designs was carefully tailored to fulfill specific functions vital to the efficacy of our biosensor.

Furthermore, we diligently pursued the enhancement and refinement of our constructs through a process of iterative improvements. Key considerations encompassed the optimization of promoter strengths, ensuring the precise normalization of reporter expression, and the implementation of measures to prevent undesirable recombination events. Additionally, we explored innovative phagemid usage for targeted Dispersin B delivery, highlighting our forward-thinking approach and adaptability in biofilm treatment.This approach underscored our commitment to crafting constructs that not only functioned effectively but did so with a high degree of precision and reliability.

Crucially, the design of our project was underpinned by a modular framework, carefully engineered to enable the seamless replacement of modules. This inherent modularity opens the door to future adaptations, underlining our commitment to a flexible and versatile biosensor that can readily evolve to meet emerging challenges and opportunities.

Figure 9: An overview of the Bye-O-Film wet lab journey. Created using the resources from Slidesgo and Freepik.

We continued with the intricate and dedicated process of creating and testing our genetic constructs. Throughout this journey, our focus remained steadfast on our goal: engineering a biosensor capable of detecting biofilm formation on medical implants. This story is one of determination and a deep commitment to our mission.

In the early days, our primary focus was on laying the foundation for our ambitious project. We took great care to ensure that our laboratory was well-equipped with the necessary materials and sterilized tools. These initial steps set the stage for the challenges that lay ahead.

Our journey continued with the critical Interlab calibration protocol, a vital step in ensuring the accuracy of our measurements. Simultaneously, we completed Experiment 2 of Interlabs, contributing our results to the broader iGEM community. Our approach was marked by diligent planning and attention to detail, as seen in the careful acquisition of plasmids and genetic components. We then turned our attention to the strategic design of the cloning process for our fluorescent biosensor plasmid—a pivotal milestone. The ensuing weeks brought a flurry of PCR and cloning activities, each step contributing to the complexity of our project. PCR was performed for various genetic components, and each presented its unique challenges. An initial setback with chemically competent cells prompted us to carefully re-evaluate our experiments. We conducted PCR cleanups, colony PCR, and minipreps to ensure the quality of our samples, underscoring our commitment to precision. Our journey continued with PCR and cloning activities, specifically related to the luciferase plasmid, promoter, and constructs involving GFP and RFP. To ensure the success of our experiments, we meticulously prepared samples for sequencing, seeking validation for the correctness of our constructs. Throughout, our discussions with our supervisors highlighted our adaptability to the evolving needs of our project. In addition, we isolated and concentrated GFP and RFP from our constructs to be tested with our electronic device.

In the ensuing weeks, we encountered challenges, particularly concerning the assembly of the CUG promoter. Despite these setbacks, we made multiple attempts to address the issues, including the strategic use of sequencing to pinpoint the root causes. Eventually switching to Golden Gate assembly and designing new sets of primers. All in all, with the iterative process of troubleshooting and changing our strategy, we have demonstrated our adaptability and problem-solving skills. In the final phase, our unwavering commitment, resilience, and relentless pursuit of our goals were evident. Challenges were met with determination, and we refined our methods through descriptive documentation, moving closer to our goal: engineering a successful biofilm detection biosensor. You can find more detailed information on our [notebook] and [experiments] pages. You can also see the overview of our timeline of activities in Figure 12.

Figure 12: Overview of the Bye-O-Film project timeline, detailing the wet lab and engineering process, focusing on the testing phase. The figure shows color-coded sequence of events against the week timeline, including a legend with more details of activities.

Our iGEM project has been a remarkable journey filled with valuable outcomes, profound learning, and significant achievements. As we reflect on this endeavor, we find that it has yielded several key results that define our success and growth.

Integration and Improvement have been the cornerstones of our project. We successfully integrated a multitude of constructs, culminating in the creation of a functional biosensor and treatment system. This achievement is a testament to our ability to blend diverse elements seamlessly, emphasizing the modularity of our design. Our project taught us the importance of adaptability, allowing us to replace or enhance components as needed, ultimately enhancing our biosensor's versatility and performance.

Our project's forward-looking perspective is embodied in our consideration of Future Directions. We recognize that our current achievements are only the beginning. By exploring the incorporation of our final product into the biosensor's genome using CRISPR technology, we anticipate improved biocontainment and system stability. This forward-thinking approach aligns with our commitment to constant growth and refinement.

Throughout this transformative journey, the principles of Modularity and Adaptability have been our guiding stars. Modularity has enabled us to approach our project with flexibility, tailoring our biosensor's sensor, reporter, or effector components to suit various applications. Adaptability ensures that we are prepared to address evolving needs and embrace future developments with open arms.

Figure 14: A collection of images representing testing phase of our engineering cycle. Depicted are the following moments in our journey: 1. Thomas carrying the ordered materials that we have received. 2. Thomas, Roos and Ingrid working in the lab on both desk research and experietns. 3. Our lab fridges filled with sticky notes with daily tasks. 4. The contaminated glassware we collected in only one week of work. 5. The collection of papers containing notes, protocols, instructions, calculations, and much more, collected during the last month of our projec.

Our hardware development was fueled by the aspiration to address a significant need in synthetic biology. We undertook the formidable task of creating an electronic biosensor capable of detecting light signals, even biological ones, such as Green Fluorescent Protein (GFP). This hardware not only represents an innovative solution but also offers practicality and affordability. It meets the demands of various users, from students embarking on scientific journeys to seasoned researchers pushing the boundaries of synthetic biology. Our hardware's modular design ensures it's adaptable for diverse purposes, whether for educational endeavors, lab experiments, or broader scientific applications. The compact and portable nature of our hardware makes it an invaluable tool, seamlessly integrating into both laboratory and remote scientific work.

The pinnacle of our hardware development was marked by a compelling proof of concept. This tangible demonstration not only illustrated the practical utility of our hardware but also showcased its functionality. Our comprehensive documentation of this proof of concept serves as compelling evidence of our device's capability to detect fluorescent proteins effectively. It reaffirmed our commitment to delivering a versatile hardware solution that could significantly elevate synthetic biology experiments and biofilm detection methods. You can read more about the details of our design on the [electronics] page. We have incorporated iGEM engineering principles in this aspect of our project too, and below we detail and discuss the events furing the different engineering phases. As a recap, we bring back Figure 4.

Figure 4: (again) Visual overview of the engineering process, based on the iGEM resources. Created using the resources from Slidesgo and Freepik.

1. Design

In our project, we aimed to create a complementary electronic device to our biological component of the biosensor. We determined, based on biological design, that we wanted our sensor to sense bioluminescence or fluorescence and to transmit real-time data to a mobile app. We started developing a proof-of-concept prototype, encompassing sensing light signals through an optical fiber, directing them to a photomultiplier tube, and processing the data with an Arduino microcontroller.

2. Build

In the build phase, we aimed to construct the physical components of our biofilm detection system. This phase involved selecting and sourcing materials, such as LED lights, optical fiber cables, phototransistors, and other electronic components. We built the system to detect light emissions from fluorescent proteins, specifically green fluorescent protein (GFP) and red fluorescent protein (mCherry/RFP). These proteins emitted light when exposed to specific wavelengths, and our device was designed to capture and amplify these signals. In the process, we consulted with experts and stakeholders to ensure optimal design.

3. Test

Testing was a critical part of our project. This phase involved conducting experiments to measure light emissions under various conditions, including different optical fiber cable lengths and concentrations of GFP and RFP. We used Arduino code to process the data and assess the system's ability to detect biofilm-related signals. Through these tests, we aimed to validate the performance of our biofilm detection system and gather quantitative data to analyze.

4. Learn

The "learn" phase of our project was focused on analyzing the results obtained during testing and using that data to refine our models and design parameters. We looked for any discrepancies between the desired and observed system functions, often with a focus on quantitative data analysis. We also considered various challenges and questions related to our system's setup and functionality. These included issues such as LED intensity, calibration, external light interference, and the need for biocompatible components in the final prototype.

Furthermore, we outlined future improvements, such as standardized ports, precision cutting equipment, a model to simulate the optical properties of human tissue, and the incorporation of a photomultiplier tube to enhance light sensitivity.

Our hardware development isn't just about solving a problem; it's about revolutionizing the way biofilm detection and monitoring occur on medical implants. With further refinement and development, our hardware holds the potential to become even more cost-effective, modular, and readily manufacturable. Future iGEM teams can harness our design, materials list, and instructions to construct their biosensors, tailoring them to their unique research requirements. As a team, we've laid the groundwork for future advancements in synthetic biology hardware, firmly believing in the transformative potential of our contributions.

Our electronics experience was not without its share of considerations and challenges, including LED intensity, calibration, external light interference, and safety. Looking ahead, we envision fine-tuning these aspects, and conducting experiments with improved control parameters to enhance hardware reliability. Future iterations might encompass standardized ports, precision equipment for fiber optic cable preparation, models simulating human tissue optical properties, and integrating a photomultiplier tube for heightened light sensitivity.

In conclusion, our dedicated hardware development efforts are underpinned by a profound commitment to addressing real needs in synthetic biology, engaging proactively with users, and providing comprehensive documentation to ensure reproducibility. Our vision is for this hardware to stand as a transformative asset for future synthetic biology projects, and we eagerly anticipate its evolution and its contributions to the iGEM community and beyond.

Figure 16: A collection of images showcasing the design cycle for the hardware. We once again began with a brainstorming session to design the electronic sensor. (left) Then we continued to code the arduino and construct the hardware, with Dyllan and Radu working on it through the nights (middle). Lastly, we had multiple sessions of testing the hardware and adjusting the design and experiment based on the sensitivity of the sensor (right). Upon issues with the sensor, we troubleshooted the electronic design as well as the code, fixed the issue and continued to test.