iGEM 2025 — Team korea-hs

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# Notebook

* [Team Meetings](#nb-team-meetings)
* [Interviews & Calls](#nb-interviews-calls)
* [Human Practices](#nb-human-practices)
* [Sustainable Development](#nb-sdg)
* [Wiki & Deliverables](#nb-wiki-deliverables)
* [Wet Lab Sessions](#nb-wetlab)

### Team Meetings

| Date | Plan/Agenda |
| --- | --- |
| 03/09/2025 | **1st Whole Team Meeting (Virtual)**  The team held its first orientation to establish communication platforms and share initial resources. Members were introduced to action items for joining iGEM platforms and preparing for future work. |
| 03/16/2025 | **2nd Whole Team Meeting (Virtual)**  The team reviewed onboarding tasks and continued orientation by finalizing the member roster and discussing leadership elections. We were also introduced to our research focus on fruit peel degradation (pectin/PG) along with award criteria and competition requirements. |
| 03/23/2025 | **3rd Whole Team Meeting (Virtual)**  The team continued onboarding by reviewing readings and preparing for upcoming journal club deadlines. We also discussed the leadership election process and learned more about our research direction. Additionally, team members were officially assigned to subgroups (Wiki, SDG, Human Practices, and Experiments). |
| 03/30/2025 | **4th Whole Team Meeting (Virtual)**  Team leader, co-leader, and sub-team leader candidates presented their speeches, followed by the voting process. This meeting marked the transition into defining the team’s leadership structure. |
| 04/06/2025 | **5th Whole Team Meeting (Virtual)**  The team reviewed award strategies and judging rubrics for special awards. Professor Lee also delivered a lecture on polygalacturonase (PG), a pectinase enzyme central to our research project. |
| 04/13/2025 | **6th Whole Team Meeting (Virtual)**  The team reviewed progress and continued refining competition strategies and upcoming plans. Professor Lee provided another lecture on the research topic to deepen understanding. |
| 04/20/2025 | **7th Whole Team Meeting (Virtual)**  Competition Strategies & Research Lecture: The team continued discussions on competition strategies and upcoming plans. Professor Lee also gave a further lecture expanding on our research topic. |
| 04/27/2025 | **8th Whole Team Meeting (Virtual)**  The team checked progress across subgroups and discussed near-term deliverables. Members coordinated on timelines and shared ideas for upcoming tasks to keep the project on track. |
| 05/04/2025 | **9th Whole Team Meeting (Virtual)**  The meeting focused on reviewing ongoing work and clarifying responsibilities. The team also outlined next steps and prepared for upcoming deadlines. The team held follow-up discussions to monitor overall progress and adjust plans as needed. Members also talked about upcoming activities and shared feedback on current work. |
| 05/13/2025 | **10th Whole Team Meeting (Virtual)**  The team shared updates on subgroup work and coordinated next steps. Members discussed short-term priorities and upcoming deadlines. |
| 05/20/2025 | **11th Whole Team Meeting (Virtual)**  The meeting focused on progress updates and preparations for upcoming activities. The team also outlined tasks to keep all groups aligned. |
| 05/25/2025 | **12th Whole Team Meeting (Virtual)**  The team focused on onboarding tasks, including wet lab sign-ups, group-specific assignments, and preparation for the upcoming journal club. Members were reminded to stay on schedule. |
| 06/01/2025 | **13th Whole Team Meeting (Virtual)**  The team continued onboarding by reviewing wet lab sign-ups and team-specific responsibilities. Members, in assigned groups, also prepared for the upcoming Journal Club #2 in Week 4. |
| 06/15/2025 | **14th Whole Team Meeting (Virtual)**  The session featured Journal Club #2 presentations (accompanied by a discussion session) followed by a short team progress check. |
| 06/22/2025 | **15th Whole Team Meeting (Virtual)**  The team reviewed team-specific responsibilities and prepared for the upcoming in-person meeting on June 28. Further plans and action items for in-person activities were also discussed. |
| 06/28/2025 | **16th Whole Team Meeting (In-person)**  The team held its first in-person meeting with a competition lecture, professional individual photoshoot, and group photoshoot. Members also gathered in subgroups for focused discussions and planning. |
| 07/05/2025 | **17th Whole Team Meeting (Virtual)**  The team used this session to share creative ideas for outreach and communication. Members also discussed strategies to make project deliverables more engaging and accessible. |
| 07/12/2025 | **18th Whole Team Meeting (Virtual)**  The meeting focused on subgroup coordination and project planning. Action points were assigned to ensure steady progress across all areas. |
| 07/19/2025 | **19th Whole Team Meeting (Virtual)**  The meeting focused on checking overall progress and aligning subgroup efforts. Members shared updates and discussed short-term goals. |
| 07/26/2025 | **20th Whole Team Meeting (In-person)**  The meeting served as a study hall for subgroups (SDG, Human Practices) to work on their tasks and further interviews. Members also caught up on team-specific deadlines and progress updates. |
| 08/03/2025 | **21th Whole Team Meeting (Virtual)**  The team held follow-up discussions on ongoing projects and coordinated next steps. Progress across subgroups was briefly reviewed. |
| 08/10/2025 | **22th Whole Team Meeting (Virtual)**  The team reviewed subgroup progress and upcoming deadlines. Members were also introduced to details about the Jamboree in Paris. |
| 08/17/2025 | **23th Whole Team Meeting (Virtual)**  The meeting centered on upcoming deadlines and refining tasks. Members also discussed ways to stay on track with project milestones. |
| 08/24/2025 | **24th Whole Team Meeting (Virtual)**  This meeting centered on reviewing ongoing experimental work and integrating it with wiki and design efforts. Subgroups shared updates to ensure consistency across all outputs. |
| 08/31/2025 | **25th Whole Team Meeting (Virtual)**  The session was used as a working meeting, where subgroups actively edited drafts, organized data, and coordinated on immediate tasks. Quick check-ins kept the pace efficient. |
| 09/07/2025 | **26th Whole Team Meeting (Virtual)**  The team discussed mid-term goals and how to balance experimental, wiki, and outreach responsibilities. Members also identified risks and brainstormed backup plans. |
| 09/14/2025 | **27th Whole Team Meeting (Virtual)**  This meeting highlighted communication and public engagement strategies. The team talked about social media, external outreach, and preparing to showcase their project story effectively. |

### Interviews & Calls

| Date | Mode | Plan/Agenda |
| --- | --- | --- |
| 06/02/2025 | (Virtual) | **Yun Seongjun, PhD Interview**  Interview Summary: discussed the potential benefits and limitations of using genetically engineered bacteria for food waste decomposition, highlighting safety concerns, public perception of GMOs, and regulatory challenges. |
| 06/03/2025 | (Virtual) | **Robert L Paarlberg Interview**  Interview Summary: Public resistance to GMOs stems from lack of direct benefits and fear, while CRISPR is seen as more acceptable due to its precision and absence of foreign DNA transfer. Changing public opinion is difficult, so companies should focus on clear benefits and safety, as environmental release of engineered bacteria could raise concerns similar to past controversies. |
| 06/06/2025 | (Virtual) | **Jae Sung Cho Interview**  Interview Summary: Dr. Cho advised optimizing bacterial growth conditions such as media, temperature, and methylation requirements for successful transformation, and emphasized using literature from related species for guidance. He recommended clear problem framing, strong biosafety measures like non-natural amino acids or inducible systems, and public engagement to address GMO concerns, noting that negative perceptions stem from early controversies and corporate framing. |
| 06/09/2025 | (Virtual) | **Sheila Jasanoff Interview**  Interview Summary: Experts advised grounding the project in strong literature, transparent risk-benefit assessments, and public engagement to address both safety and ethical concerns. They stressed demonstrating the urgency of food waste, planning for unintended consequences, and ensuring collaboration between scientists, policymakers, and society to align innovation with trust and democratic values. |
| 06/28/2025 | (In-Person) | **Citizen Surveys & Interviews**  Interview Summary: Conducted interviews to collect various thoughts on food wastes (related with our topic). |
| 06/29/2025 | (Virtual) | **Jangchung-dong Wangjokbal Interview**  Interview Summary: The project began in 2021 as part of Korea’s Smart Eco Factory initiative, aiming to achieve zero waste and integrate renewable energy in food manufacturing. While initial plans to use microbial fermentation for waste recycling faced technical, regulatory, and cost barriers, the focus shifted to heat-based treatment; for future commercialization of biotech solutions, key factors include cost competitiveness, regulatory compliance, reusability of microbes, and ensuring profitability for long-term sustainability. |
| 07/18/2025 | (In-Person) | **Exhibition Corporate Fair Interviews**  API Tech: Interviews to discuss product and project-related questions.  Lock & Greenholic Co., Ltd: Interviewed about consumer attitudes and behaviors regarding fruit peel waste disposal, their awareness and perception of genetically modified enzymes, and their openness to using microbial technology for waste decomposition  PhytorySearch: The team interviewed industry experts and consumers to understand perspectives on fruit peel waste management, public attitudes toward genetically modified and microbe-based decomposition technologies, and the technical, regulatory, and ethical considerations involved in implementing such solutions.  Ttogabi Yoonsseu: The interview explored how participants handle food waste, their willingness to use fruit peel decomposition via probiotics, and, from a food producer’s perspective, what standards or regulations might apply to upcycling processes intended for use in food products.  Korea Agricultural Policy Insurance & Finance Service (APFS): The interview focused on how the APFS evaluates inventions or technologies that can enhance farmers’ long-term financial well-being, the criteria used to assess whether new agricultural technologies provide financial stability or pose risks, and recommended data-driven approaches for student research teams to support marginalized farming communities, such as elderly or small-scale farmers.  Korea Agricultural Policy Insurance & Finance Service (APFS): The discussion explored which technologies or inventions are most likely to enhance farmers’ long-term financial welfare and what criteria or benchmarks companies consider when deciding to invest in agricultural insurance or related programs.  Cotegreen Agricultural Corporation: The interview addressed how technologies for efficiently processing agricultural waste can support eco-friendly farming and fertilizer development, the validation and testing steps required to translate lab-developed technologies to real-world farm applications, and the potential for byproducts from food waste decomposition to be safely used as fertilizers or soil amendments.  Baekkob Food: The interview focused on how the company manages food waste, such as fruit and vegetable peels, generated during production, and which factors—like cost and operational feasibility—they prioritize when implementing waste-reduction technologies.  Viva Nature AgriScience: The interview explored the company’s perspective on how synthetic biology–based waste-processing technologies might impact crop environments, and whether interactions between soil microbes and seed growth are studied or experimentally considered when developing new plant varieties.  Brocos Co., Ltd: The interview focused on the motivation for developing healthy products from ingredients like broccoli stems, how the public perceives functional foods derived from nontraditional plant parts, and whether there has been any consumer resistance to products made from parts of the plant that would otherwise be discarded.  Korea Agency of Agri-Food Science & Technology Planning: The interview addressed how new agricultural technologies are evaluated for their environmental impact and what ethical considerations researchers should account for when designing synthetic biology–based projects.  Modooang Winery: The question asks whether the respondent disposes of grape skins directly into the soil.  GayaBio: The interview explored how the company approaches carbon neutrality in livestock farming, the role of animal welfare in its R&D processes, and the contribution of the company to shaping the future of sustainable agriculture and economy.   LuxBiome: The interview explored the company’s strategies for achieving carbon neutrality in livestock farming, the role of animal welfare in R&D, its contribution to shaping sustainable agriculture, and how human microbiome research, bioinformatics, and systems biology are applied in developing practical health solutions and products.  Entec Bio-S: The interview focused on how Entech Bios designs plants or microbes using synthetic biology to improve biomass conversion efficiency, the company’s unique strengths compared to domestic and international competitors in biomass technologies, and its goals or strategies for expanding or exporting these technologies abroad.  Phyto Research: The company explained that it uses approaches to optimize growing conditions and increase crop yields, and the interview explored the reasons behind adopting this systematic method, such as improving efficiency, sustainability, and overall production quality.  Food Connect Co., Ltd: The interview asked whether there are specific standards that microbial upcycling processes must meet to ensure the safety of food/beverages, and whether producers perceive public awareness and acceptance of such processes as having improved. |
| 07/26/2025 | (In-Person) | **Survey of Merchants at Gwangjang Market**  Fruit Vendor / Fruit Seller: The interview asked what the concerns are when disposing of fruit peels and whether the participant would be willing to use a bacterial method that could break down the peels and convert them into fertilizer if such a technology were developed.  Ssuk’s Clerk: The interview explored how participants handle fruit peel waste at home or in stores, the costs involved, their familiarity with and attitudes toward genetically modified enzymes, whether they consider current waste disposal methods eco-friendly, and their willingness to use microbial technologies for decomposing fruit peels.  Apple-Watermelon Juice Vendor: The interview asked what concerns participants have when disposing of fruit peels from juice products, the costs associated with disposal, their opinion on technologies that could decompose the peels, and whether they would feel uncomfortable using genetically modified enzymes for this purpose.  K-Snacks Art Food Owner: The interview explored participants’ concerns when disposing of fruit peels, including issues like pests, their attitudes toward using genetically modified enzymes for decomposition, whether they consider current disposal methods eco-friendly, their willingness to adopt microbial decomposition technologies, and any additional features they would like such technologies to include. |

### Human Practices

| Date | Mode | Plan/Agenda |
| --- | --- | --- |
| 03/29/2025 | (Virtual) | Introduction to Human Practices (What is HP, Integrated HP). |
| 04/05/2025 | (Virtual) | Identified stakeholder cohorts; discussed outreach approaches. |
| 04/12/2025 | (Virtual) | Sourced 30 stakeholders per member across Socioeconomic, Environmental, Ethical/Awareness, Scientific/Technological. |
| 04/19/2025 | (Virtual) | Continued stakeholder sourcing and individual feedback. |
| 05/25/2025 | (Virtual) | Drafted cold email template; sent outreach emails to stakeholders. |
| 06/01/2025 | (Virtual) | Prepared interview questionnaires for Yun Seongjun, PhD. |
| 06/08/2025 | (Virtual) | Finished stakeholder interviews and prepared for further interviews. |
| 06/15/2025 | (Virtual) | Edited questionnaire for Jangchung-dong Wangjokbal; organized interview status; prepared in-person initiatives (6/28). |
| 06/19/2025 | (Virtual) | Organized interview status; prepared in-person initiatives (6/28); organized Google Drive. |
| 06/22/2025 | (Virtual) | Prepared for 7/18 Exhibition and 7/26 Gwangjang Market; organized Google Drive. |
| 07/06/2025 | (Virtual) | Finished company descriptions. |
| 07/12/2025 | (Virtual) | Continued prep for 7/18 & 7/26 initiatives; organized Google Drive. |
| 07/20/2025 | (Virtual) | Prepared questionnaires for 7/26 Gwangjang Market; organized Google Drive. |
| 08/03/2025 | (Virtual) | Finished interview transcriptions. |
| 08/10/2025 | (Virtual) | Organized interview status & Google Drive; began Integrated HP. |
| 08/18/2025 | (Virtual) | Began Integrated HP; created section docs and drafts. |
| 08/24/2025 | (Virtual) | Continued Integrated HP. |
| 08/31/2025 | (Virtual) | Continued Integrated HP. |
| 09/07/2025 | (Virtual) | Integrated HP framework; Instagram posts; created shared progress doc; added diagrams. |

### Sustainable Development

| Date | Mode | Plan/Agenda |
| --- | --- | --- |
| 03/29/2025 | (Virtual) | The first SDG meeting includes an icebreaker, a discussion on SDGs and stakeholders, and short lectures on judging criteria and past examples. The team also introduces its project: engineering bacteria to produce the PG enzyme to speed up fruit peel degradation for sustainability. |
| 04/05/2025 | (Virtual) | The team reviewed the basics of SDG alignment and shared reflections on why their project matters in a sustainability context. Members also began considering which stakeholder groups would be most relevant. |
| 04/12/2025 | (Virtual) | The meeting finalized stakeholder groups and outreach methods. Each SDG lead will contact relevant organizations (e.g., food producers, public health agencies, environmental companies, and educational institutions) using websites, scholarly sources, LinkedIn, and direct emails. Members are tasked with sourcing 30 stakeholders for their assigned goal. |
| 04/19/2025 | (Virtual) | The meeting focused on checking progress with assigned stakeholder research. Members exchanged early findings and discussed challenges in identifying reliable points of contact. |
| 04/26/2025 | (Virtual) | The team continued with stakeholder outreach planning, emphasizing clear communication strategies. Discussions also touched on how to connect project goals with broader SDG themes. |
| 05/03/2025 | (Virtual) | This meeting was used to share updates from tasks and refine the overall outreach approach. Members highlighted the importance of documenting progress for future use. |
| 05/10/2025 | (Virtual) | The group discussed potential interviewees and brainstormed questions for interviews. Members also shared ideas for simple outreach materials. |
| 05/17/2025 | (Virtual) | The meeting centered on preparing for upcoming deadlines and ensuring each member stayed on track. Members agreed to finalize initial drafts of outreach content before the next session. |
| 05/25/2025 | (Virtual) | SDG Meeting 4 focused on checking in with individual tasks from Meeting 3 and discussing future directions for the project after May 20. |
| 06/07/2025 | (Virtual) | The team reviewed outreach responses received so far and discussed how to follow up. The conversation also included ideas for how to integrate stakeholder feedback into project planning. |
| 06/14/2025 | (Virtual) | The meeting focused on project planning, aligning with SDG goals, and preparing for stakeholder outreach. It concluded with action steps to continue engagement and move the project forward. |
| 06/21/2025 | (Virtual) | The meeting centered on planning SDG-related activities to expand outreach and education. The team decided to create an interactive STEM lesson for YouTube highlighting food waste, biotechnology, and the SDGs, and to design visual materials like posters or infographics illustrating the lifecycle of food waste and sustainable enzyme solutions. |
| 06/28/2025 | (Virtual) | The team reviewed ongoing progress and prepared for the upcoming orientation session. Members shared updates from their subgroups and aligned on next steps to keep work consistent. |
| 07/05/2025 | (Virtual) | The meeting centered on final preparations for orientation activities. Members discussed logistics for distributing materials and how to introduce the project effectively. |
| 07/14/2025 | (Virtual) | The meeting focused on preparing activities for the upcoming in-person orientation, including a survey and pamphlet distribution. Members checked progress and set action items such as interview preparation, pamphlet design, survey setup, and brainstorming social media content. |
| 07/21/2025 | (Virtual) | The meeting focused on preparing for the upcoming expo, including brainstorming ideas for an interactive lesson video. The team also planned future actions and activities connected to key SDGs: Responsible Consumption and Production, Climate Action, Sustainable Cities and Communities, and Quality Education. |
| 07/28/2025 | (Virtual) | The meeting was dedicated to wrapping up unfinished tasks from the previous week, starting work on the interactive lesson video, and selecting activities from the brainstormed ideas to move forward with. |
| 08/04/2025 | (Virtual) | The team checked in on progress with the interactive lesson video and other outreach tasks. Discussions also included adjusting the timeline to balance multiple deadlines. |
| 08/11/2025 | (Virtual) | The meeting focused on completing remaining tasks, reviewing stakeholder responses, and beginning work on the top-voted activity. Members also planned to upload videos and photos from the recent in-person visit to Google Drive. |
| 08/18/2025 | (Virtual) | The meeting centered on completing pending tasks, finalizing slides for the interactive lesson video, and beginning recording. The team also worked on drafting a script for the fruit peel visual and started developing slides for that activity. |
| 08/26/2025 | (Virtual) | The meeting focused on recording assigned sections of the project and beginning the editing process. |
| 09/01/2025 | (Virtual) | The meeting focused on checking progress from previous tasks, continuing work on projects, and discussing actions and activities. |
| 09/08/2025 | (Virtual) | The team provided updates on ongoing editing and outreach tasks. Members reflected on lessons learned from recent activities and adjusted future plans accordingly. |
| 09/15/2025 | (Virtual) | The final meeting emphasized wrapping up deliverables and ensuring everything was prepared for submission. |

### Wiki & Deliverables

| Date | Mode | Plan/Agenda |
| --- | --- | --- |
| 03/30/2025 | (Virtual) | The team held an icebreaker session and introduced members to each other. Roles and responsibilities were briefly outlined, giving everyone an overview of the deliverables team’s tasks. |
| 04/06/2025 | (Virtual) | The meeting focused on discussing the team’s role in the overall project. Members explored how media & branding would contribute to goals. |
| 04/13/2025 | (Virtual) | Members shared personal strengths and creative skills, deciding how they might apply to logo, apparel, and media work. The team also began discussing early competition strategies. |
| 04/20/2025 | (Virtual) | The group reviewed expectations for Wiki & Deliverables and talked about timelines. Members brainstormed ideas for branding concepts. |
| 04/27/2025 | (Virtual) | This session centered on setting communication norms and working platforms. Members also reflected on how to stay organized. |
| 05/04/2025 | (Virtual) | The team kicked off design brainstorming for the t-shirt, sharing sketches and references. Members compared concepts and styles for consistency. |
| 05/11/2025 | (Virtual) | Progress on the t-shirt design was shared and critiqued. The group discussed printing options, and how the design could reflect the theme. |
| 05/18/2025 | (Virtual) | The final t-shirt design was completed. Members confirmed vendor details and prepared the order list. |
| 05/25/2025 | (Virtual) | The meeting was devoted to sending out the t-shirt order. The group also began casually brainstorming what the team outline might include. |
| 06/01/2025 | (Virtual) | Members drafted the team outline, sketching out structure for wiki content and identifying what visuals would be needed. |
| 06/08/2025 | (Virtual) | The first round of promotion video script brainstorming began. The team discussed narrative flow, tone, and what story they wanted to tell. |
| 06/15/2025 | (Virtual) | Work continued on the script, with the second draft being developed. Members refined the storyline and emphasized clarity for audiences. |
| 06/22/2025 | (Virtual) | The script reached its third round of editing, with feedback from across the team incorporated. Plans for visuals and graphics were outlined. |
| 06/29/2025 | (Virtual) | The script was finalized for submission. Attention shifted toward planning for actual video production. |
| 07/06/2025 | (Virtual) | The team began recording and editing the first cut of the video. Members reviewed initial footage and aligned it with the script. |
| 07/13/2025 | (Virtual) | The second cut of the video was refined with smoother transitions and graphics. Members provided targeted feedback on pacing and style. |
| 07/20/2025 | (Virtual) | The third cut of the video was completed, focusing on audio balance and subtitles. The team prepared the final round of revisions. |
| 07/27/2025 | (Virtual) | The final promotion video was completed and submitted. The team reflected on takeaways and began shifting to wiki theme discussions. |
| 08/03/2025 | (Virtual) | The wiki theme was finalized, and members discussed color palettes, layouts, and coherence. Responsibilities were divided for sections. |
| 08/10/2025 | (Virtual) | Commenting on the wiki theme took place. Members shared critiques on readability and consistency. |
| 08/17/2025 | (Virtual) | The meeting centered on wrapping up ongoing wiki work while beginning to shift focus toward the notebook. Members discussed how to organize documentation and what materials would be needed. |
| 08/24/2025 | (Virtual) | The team continued with planning for the notebook and reviewed progress across other deliverables. Discussions touched on formatting, visuals, and keeping deadlines manageable. |
| 08/31/2025 | (Virtual) | Notebook drafting began, with members collecting experimental and outreach notes. Work was divided for efficient progress. |
| 09/07/2025 | (Virtual) | The notebook draft was reviewed, with edits for clarity and formatting. |
| 09/14/2025 | (Virtual) | The final notebook edits were made. The team also finalized hoodie and booth design concepts, ensuring that visuals were complete. |

### Wet Lab Sessions

| Date | Mode | Session | Activities |
| --- | --- | --- | --- |
| 07/06/2025 | (In-Person) | Group 1: Session 1 | Practiced micropipette use (5 trials with water).  Added 1 µL DNA into E. coli solutions and mixed.  Heat shock: 1 min 30 sec at 46 °C → ice incubation 5 min.  Added 100 µL LB media → 30 min recovery at 37 °C; shaking incubator (200 rpm, 30+ min).  Prepared LB agar + antibiotics; poured plates; plated 100 µL; incubated 37 °C for 1 day. |
| 07/13/2025 | (In-Person) | Group 1: Session 2 | Transferred colonies to tubes; centrifuged E. coli; discarded supernatant.  EX Pure Plasmid Kit extraction (resuspend, lyse, neutralize, centrifuge).  DNA binding column → wash → elute; measured plasmid purity (2 µL samples, ED control). |
| 07/20/2025 | (In-Person) | Group 2: Session 1 | *Pre-Lab:* Micropipette practice.  *Protein extraction:* Centrifuge L. reuteri samples; lysis buffer; vortex; sonication; separate debris; prepare 4 tubes (+/− plasmid cells/solutions); add SDS 1×.  *Electrophoresis:* Fill apparatus; load marker (3 µL) + 4 samples; run 160 V, 0.03 A, 40 min.  *Staining:* Remove gel; cut wells; stain with Coomassie on nutating mixer. |
| 07/27/2025 | (In-Person) | Group 2: Session 2 | *Protein extraction:* Centrifuge (~22 °C, 3000 rpm, 3 min); collect supernatants; add lysis buffer; sonicate 15 min (amp 1/40%, pulse 3 sec/3).  *Bradford assay:* Prepare BSA standards S0–S5; Bradford dye; 96-well: 195 µL dye + 5 µL of standards and +/− samples; incubate 3 min; read 595 nm. |
| 08/03/2025 | (In-Person) | Group 3: Session 1 | *Pre-Lab:* Pipetting practice.  *DNS reagent:* 0.5 g DNS, 0.8 g NaOH, 15 g sodium potassium tartrate, water to 50 mL.  *Standard curve:* 6 D-galacturonic acid standards (stock) of varying concentrations.  *Pectin assay:* 8 tubes (4 plasmid / 4 no plasmid): 4.5 mL 1% pectin + 0.5 mL supernatant; incubate 37 °C; collect 1 mL; DNS assay. |
| 08/10/2025 | (In-Person) | Group 3: Session 2 | *Fruit peels:* Prepare peels (Korean pear, watermelon, banana, kiwi, tomato); grind with 50 mL water; centrifuge.  *Enzyme reaction:* For each fruit: 300 µL extract + 200 µL plasmid solution (+/−); vortex. DNS tubes: 50 µL DNS + 50 µL sample; heat 100 °C, 5 min.  *Absorbance:* 80 µL per well in 96-well; DNS-only control; read 540 nm. |

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# Engineering

* [Engineering Cycle #1: Design](#eng-design)
* [Engineering Cycle #2: Build](#eng-build)
* [Engineering Cycle #3: Test](#eng-test)
* [Engineering Cycle #4: Learn](#eng-learn)

### Engineering Cycle #1: Design

#### Design Purpose

To achieve efficient fruit peel degradation, we designed a recombinant construct enabling *Lactobacillus reuteri* to secrete the polygalacturonase enzyme (pgxC). The construct was assembled in the shuttle plasmid pTRKH3, allowing stable propagation in *E. coli* and functional expression in *L. reuteri*. The design consists of the following elements:

* **Promoter (ermB promoter)** – a strong constitutive promoter ensuring robust gene expression in Gram-positive bacteria.
* **Secretion Signal (Usp45)** – a well-characterized leader peptide from *Lactococcus lactis* that directs the expressed protein into the extracellular medium.
* **Coding Sequence (pgxC)** – the polygalacturonase enzyme (48.5 kDa), responsible for cleaving the α-1,4-glycosidic linkages in pectin to release D-galacturonic acid.
* **Terminator** – native transcriptional terminator included for stability.

We registered this construct as a composite part in the iGEM Registry:

* **Part ID:** BBa\_K5000001
* **Name:** [ermB promoter + Usp45 signal peptide + pgxC]
* **Function:** Extracellular secretion of polygalacturonase for fruit peel degradation
* **Plasmid Backbone:** pTRKH3 (erythromycin resistance marker)

![Part ID: BBa_K5000001 with ermB promoter + Usp45 signal peptide + pgxC.](https://static.igem.wiki/teams/5773/engineering/eengineering-figure1.avif)

(Figure 1. Part ID: BBa\_K5000001 with ermB promoter + Usp45 signal peptide + pgxC.)

![Sequence design of composite part BBa_K5000001.](https://static.igem.wiki/teams/5773/engineering/eengineering-figure2.avif)

(Figure 2. Sequence design of composite part BBa\_K5000001.)

The construct consists of the ermB constitutive promoter, followed by an EcoRI restriction site introduced at the 5′ end for cloning, the Usp45 secretion signal peptide, and the codon-optimized pgxC coding sequence. A downstream transcriptional terminator ensures stable expression. This design allows *Lactobacillus reuteri* to secrete active polygalacturonase for efficient fruit peel degradation.

![Sequence design of the 3′ region of codon-optimized pgxC.](https://static.igem.wiki/teams/5773/engineering/ee3engineering-figure3.avif)

(Figure 3. Sequence design of the 3′ region of codon-optimized pgxC.)

The terminal region of the pgxC coding sequence was engineered to include a BamHI restriction site at the 3′ end, allowing for flexible downstream cloning. Sequence alignment highlights the codon-optimized pgxC region, with annotated restriction sites (PpuMI, PciI, SexAI) for verification and modular assembly. This design ensures that pgxC can be efficiently integrated and expressed in the final plasmid construct for extracellular polygalacturonase secretion.

![Predicted 3D structure of engineered pgxC fused with USP45 signal peptide.](https://static.igem.wiki/teams/5773/engineering/ee3engineering-figure4.avif)

(Figure 4. Predicted 3D structure of engineered pgxC fused with USP45 signal peptide.)

The structure was modeled using the ESMFold program. The USP45 secretion signal peptide (left, red box) forms an extended flexible region, while the pgxC protein main body (right, blue box) adopts a stable β-sheet-rich fold characteristic of polygalacturonase enzymes. This fusion design facilitates extracellular secretion of the active enzyme by *Lactobacillus reuteri*.

#### Additional Notes

Besides the five new parts we added to the Registry, we added two additional parts to the Registry for engineering purposes. The additions are as follows:

* Engineered N-terminal Secretion Signal Peptide pgxC-silent PstI Scar Removal (<https://registry.igem.org/parts/bba-25cmenhz>)
* ermB Promoter with Engineered N-terminal Secretion Signal Peptide pgxC-silent PstI Scar Removal (<https://registry.igem.org/parts/bba-25txyoa2>)

*\*The pgxC signal peptide was modified by a silent PstI site removal to restore RFC 10 compatibility while maintaining the original amino acid sequence and secretion functionality.*

### Engineering Cycle #2: Build

#### Overview: Building Process

To engineer *L. reuteri* for extracellular polygalacturonase (pgxC) secretion, we constructed a recombinant plasmid based on the shuttle vector pTRKH3. The construct was designed with restriction enzyme sites to enable efficient insertion of the codon-optimized pgxC gene fused to the Usp45 secretion signal peptide under control of the ermB constitutive promoter.

**Part 1: Cloning Strategy Using Restriction Enzymes**

The codon-optimized pgxC sequence was synthesized with EcoRI (5′) and BamHI (3′) restriction sites flanking the gene to facilitate cloning into the pTRKH3 plasmid vector:

* **EcoRI site:** Positioned upstream of the promoter region, ensuring correct orientation and seamless ligation.
* **BamHI site:** Placed downstream, allowing the pgxC insert to be stably joined with the vector backbone.

The pTRKH3 plasmid was linearized with these enzymes, and the pgxC fragment was ligated in-frame with the Usp45 signal sequence. This design ensures secretion of the expressed protein into the extracellular medium.

![Plasmid map of engineered construct pTRKH3-pgxC.](https://static.igem.wiki/teams/5773/engineering/ee3engineering-figure5.avif)

(Figure 5. Plasmid map of engineered construct pTRKH3-pgxC.)

Schematic representation of the recombinant pTRKH3 plasmid (9585 bp) containing the ermB promoter, Usp45 secretion signal peptide, and the codon-optimized pgxC gene. Restriction enzyme sites EcoRI and BamHI (red boxes) were introduced to enable insertion of pgxC. The construct provides erythromycin resistance for selection in both *E. coli* and *L. reuteri*.

**Part 2: Verification of Cloning**

![Verification of pgxC gene insertion into the pTRKH3 plasmid by restriction digestion.](https://static.igem.wiki/teams/5773/engineering/ee3engineering-figure6.avif)

(Figure 6. Verification of pgxC gene insertion into the pTRKH3 plasmid by restriction digestion. Agarose gel electrophoresis confirmed successful cloning of pgxC into the pTRKH3 backbone.)

The BamHI-only digest produced a single band at ~9585 bp, corresponding to the intact recombinant plasmid. The BamHI + EcoRI double digest generated two fragments: one corresponding to the vector backbone (8238 bp) and the other to the pgxC insert (1347 bp), consistent with the expected sizes of the engineered construct.

After ligation, the recombinant plasmid was first transformed into *E. coli* DH5α for propagation. Plasmid DNA was extracted and subjected to:

1. **Restriction digestion analysis** – BamHI-only digestion produced a single band (9585 bp), while BamHI + EcoRI double digestion produced two bands (8238 bp and 1347 bp), confirming correct pgxC insertion.
2. **Agarose gel electrophoresis** – Confirmed expected fragment sizes (Figure 6).
3. **Sanger sequencing** – Verified correct orientation and codon integrity of pgxC insert.

Once verified, the recombinant plasmid pTRKH3-pgxC was introduced into *L. reuteri* by electroporation, generating the engineered strain for subsequent protein expression and functional assays.

### Engineering Cycle #3: Test

#### Rationale & Objectives

Following successful construction of the recombinant plasmid pTRKH3-pgxC, it was essential to evaluate whether the engineered *Lactobacillus reuteri* could express, secrete, and functionally utilize the pgxC enzyme. The rationale behind testing was to confirm that the designed genetic circuit not only worked at the DNA level but also translated into measurable protein production and functional pectin degradation. The specific objectives of the testing phase were:

1. To confirm expression of pgxC at the protein level by detecting the expected 48.5 kDa band using SDS-PAGE.
2. To quantify the amount of secreted protein in the engineered strain compared to controls using the Bradford assay.
3. To validate the enzymatic activity of pgxC through a DNS assay that measures release of reducing sugars (D-galacturonic acid) from pectin substrates.
4. To demonstrate functional application of the engineered strain in degrading real fruit peel biomass (orange and apple), thereby linking molecular results to practical bioconversion outcomes.

Together, these tests provide a comprehensive evaluation of the system — from protein secretion to enzymatic functionality and applied biomass degradation — ensuring that the design is effective beyond genetic assembly.

**Part 1: Protein Expression and Secretion**

![Detection of secreted pgxC protein in Lactobacillus reuteri.](https://static.igem.wiki/teams/5773/engineering/ee3engineering-figure7.avif)

(Figure 7. Detection of secreted pgxC protein in *Lactobacillus reuteri*. (Left) Illustration showing separation of cell pellet and supernatant, with pgxC proteins detected in the extracellular fraction.)

![Quantification of secreted proteins using the Bradford assay.](https://static.igem.wiki/teams/5773/engineering/ee3engineering-figure8.avif)

(Figure 8. Quantification of secreted proteins using the Bradford assay. (Top) BSA standard curve generated by measuring absorbance at 595 nm across a concentration range of 0–0.5 µg/µL. The linear regression equation (y = 0.401x + 0.091, R² = 0.94) was used to calculate protein concentrations in experimental samples. (Bottom) Protein concentrations in the culture supernatants of *Lactobacillus reuteri* with and without the pgxC plasmid. Samples with the plasmid showed a significantly higher protein concentration (0.10 ± 0.01 µg/µL) compared to the control without plasmid (0.04 ± 0.005 µg/µL), confirming enhanced extracellular protein expression.)

To verify that pgxC was expressed and secreted:

* SDS-PAGE analysis revealed a distinct protein band at ~48.5 kDa in the culture supernatant of engineered *L. reuteri*, corresponding to the expected molecular weight of pgxC. This band was absent in the plasmid-free control strain.
* Bradford assay quantified total secreted protein, showing significantly higher levels in engineered strains (0.10 µg/µL) compared to controls (0.04 µg/µL).

These results confirmed successful extracellular secretion of the engineered enzyme.

**Part 2: Enzyme Activity Validation**

![Standard curve for D-galacturonic acid quantification using the DNS assay.](https://static.igem.wiki/teams/5773/engineering/ee3engineering-figure9.avif)

(Figure 9. Standard curve for D-galacturonic acid quantification using the DNS assay. The standard curve was generated by measuring absorbance at 540 nm for increasing concentrations of D-galacturonic acid (0–1.0 mg/mL).)

![Quantification of D-galacturonic acid after 0, 12, 24, 48h incubation](https://static.igem.wiki/teams/5773/engineering/ee3engineering-figure10.avif)

(Figure 10. Quantification of D-galacturonic acid after 0, 12, 24, 48h incubation of supernatant produced from control (no pgxC) and engineered (pgxC) samples. Values are shown as mean ± SD from three replicates.)

The functional activity of pgxC was tested using the DNS (3,5-dinitrosalicylic acid) assay:

* Engineered *L. reuteri* supernatants released significantly higher amounts of D-galacturonic acid from pectin substrate than control strains.
* Absorbance at 540 nm was used to quantify reducing sugars, with engineered strains showing a ~2.5-fold increase in activity.

This demonstrated that the secreted pgxC retained enzymatic functionality.

**Part 3: Application to Fruit Peel Degradation**
To evaluate real-world utility, tests were performed using fruit peels (orange&apple):

![Comparison of D-galacturonic acid release from fruit peels](https://static.igem.wiki/teams/5773/engineering/ee3engineering-figure11.avif)

(Figure 11. Comparison of D-galacturonic acid release from fruit peels after 24 h incubation with control and engineered *L. reuteri* supernatants. DNS assay was performed to quantify reducing sugars at 540 nm in extracts of banana, kiwi, pear, tomato, and watermelon peels.)

* Homogenized fruit peel incubations – Engineered strains showed visible degradation, including liquefaction and reduction in peel particle size, compared to controls.

These results confirmed that engineered *L. reuteri* can degrade fruit peel components, validating the practical application of the construct.

### Engineering Cycle #4: Learn

Through our Build and Test phases, we reflected on the outcomes and identified strategies to optimize our construct and experimental design. The Learn phase allowed us to evaluate the strengths and limitations of our system, and to plan improvements for future iterations.

#### Learn #1: Solving Project Problems Using Synthetic Biology Tools & Experimental Techniques

We aimed to confirm whether *Lactobacillus reuteri* engineered with pTRKH3-pgxC could effectively secrete active (pgxC) and degrade fruit peel waste. Our tests showed:

* Protein secretion was achieved, confirmed by SDS-PAGE with a visible 48.5 kDa band.
* Protein yield was modest (0.10 µg/µL vs. 0.04 µg/µL in controls), indicating successful expression but suggesting room for higher secretion efficiency.
* Enzymatic activity was validated through DNS assay, which showed ~2.5-fold more reducing sugar release in engineered strains compared to controls.
* Functional fruit peel degradation occurred, but the process was slower than expected, suggesting that enzyme activity or secretion levels may not yet be optimal at large-scale.

This confirmed that our synthetic biology approach successfully addressed the core challenge — enabling pectin degradation in a GRAS probiotic — with bottlenecks in yield and efficiency.

#### Learn #2: Next Steps and Improvements for Future Design Iterations Based on these findings, we identified several improvements for the next engineering cycle:

1. **Promoter Optimization**
   Replace ermB with stronger or inducible promoters to increase protein expression while balancing host growth.
2. **Secretion Signal Variants**
   Test alternative signal peptides (SPuspA or SlpA) to enhance extracellular protein secretion compared to Usp45.
3. **Codon Optimization Refinement**
   Re-analyze codon usage in *L. reuteri* and design optimized pgxC variants to improve translation efficiency.
4. **Enzyme Engineering**
   Generate pgxC truncation variants or directed evolution mutants to improve activity and stability under acidic fruit waste conditions.
5. **Adaptive Laboratory Evolution (ALE)**
   Expose *L. reuteri* to fruit peel-rich environments to select strains with improved fitness and degradation capacity.
6. **System-level Integration**
   Explore co-expression of accessory enzymes (pectin methylesterase and or cellulases) to enhance complete biomass breakdown.

#### Summary of Learnings:

![The flow chart of Build, Design, Learn, and Test.](https://static.igem.wiki/teams/5773/engineering/ee3engineering-figure12.avif)

(Figure 12. The flow chart of Build, Design, Learn, and Test.)

Our engineered construct proved functional in secreting pgxC and initiating fruit peel degradation, validating our design. However, efficiency limitations highlighted opportunities for promoter engineering, secretion optimization, and enzyme enhancement. These insights will guide the next iteration of the Design–Build–Test–Learn cycle to achieve higher degradation efficiency and practical scalability.

* Part 1: Cloning Strategy Using Restriction Enzymes
* Part 1: Protein Expression and Secretion
* Part 2: Enzyme Activity Validation
* Part 3: Application to Fruit Peel Degradation
* Learn #2: Next Steps and Improvements for Future Design Iterations

{% endblock %}

{% set page = {"name": "experiments"} %}
{% extends "layout.html" %}
{% block page\_content %}

# Experiments

* [Overview](#exp-overview)
* [1st Wet Lab Session](#exp1)
* [2nd Wet Lab Session](#exp2)
* [3rd Wet Lab Session](#exp3)
* [4th Wet Lab Session](#exp4)
* [5th Wet Lab Session](#exp5)
* [6th Wet Lab Session](#exp6)

### Overview

Our wet lab comprised a total of six in-person sessions at Institute for Research on Innovative Science (IRIS) Lab, headquartered in Seoul, South Korea. The biosafety provisions concerning our project can be found in the Safety page. Below, we list the experimental principle, methods, and materials for each experimentation we conducted over the course of our project:

### 1st Wet Lab Session: July 6th, 2025

#### Session Agenda #1: Micropipette Practice

**Experimental Principle:** Accurate pipetting is essential for precise DNA and bacterial handling. Practicing ensures consistency and avoids contamination.

###### Materials Needed:

* Micropipette
* Pipette tips
* Test tubes
* Distilled water

###### Experimental Procedure:

1. Check that the volume dial is set to the correct volume (10 µL).
2. Firmly press the pipette into a sterile tip until it clicks in place.
3. Tip slightly below the surface of the water and press the button to the first stop.
4. Make sure the pipette is perpendicular to the liquid.
5. Slowly release the button to draw water into the tip.
6. Make sure there are no air bubbles.
7. Press the button to the first stop and expel all liquid.
8. Pour the liquid close to the container in contact.
9. Pour the liquid onto the bottom middle of the container.
10. Repeat five times per person to practice.

![Pipetting water to a small container for accuracy and precision.](https://static.igem.wiki/teams/5773/experiments/exp-figure1.avif)

Figure 1 (Pipetting water to a small container, practicing for better accuracy and precision.)

#### Session Agenda #2: DNA Introduction into E. coli

**Experimental Principle:** Introducing the plasmid DNA into E. coli cells before heat shock allows transformation during the next step.

##### Materials Needed:

* 4 test tubes with E. coli solution on ice
* Alcohol burner
* Fume hood
* Plasmid DNA (1 µL per tube)
* LB media (100 µL)
* Micropipette & sterile tips

##### Experimental Procedure:

1. Place the test tubes containing E. coli on ice in a rack.
2. The test tubes are labelled with each group name.
3. Micropipette 1 µL of the plasmid DNA into the E.coli solution for each 4 test tubes.
4. We worked at the fume hood. An alcohol burner was lighted up in the fume hood to minimize airborne contamination.
5. Make sure to tap the side of each tube with your thumb after micropipetting in order to mix the solution evenly without creating bubbles.

![Adding plasmid DNA to E. coli in a fume hood.](https://static.igem.wiki/teams/5773/experiments/exp-figure2.avif)

Figure 2 (Adding plasmid DNA into the E. coli solution inside the fume hood.)

###### Session Agenda #3: Heat Shock Transformation

**Experimental Principle:** Rapid heat shock creates temporary pores in bacterial membranes, allowing plasmid entry. LB media helps recovery and provides nutrition.

###### Materials Needed:

* Water bath (46 °C)
* 4 E. coli + DNA solution test tubes
* Ice container
* LB media (100 µL per tube)
* Micropipette & tips

###### Procedure

1. Transfer 4 tubes (E. coli + DNA) from ice into a 46 °C water bath for 1 min 30 sec.
2. Immediately return tubes to ice for 5 min recovery.
3. Pipette 100 µL of the LB (Luria-Bertani) media solution into each test tube.
4. Make sure to tap the test tube with your thumb to mix evenly.
5. Place tubes in a 37 °C incubator for 30 min to recover.

![Heat-shocking E. coli + DNA tubes in a water bath.](https://static.igem.wiki/teams/5773/experiments/exp-figure3.avif)

Figure 3 (Water bathing the E.coli + DNA tubes for heat shock.)

#### Session Agenda #4: Agar Plate Preparation

**Experimental Principle:** LB agar provides nutrients and a surface for colonies.

###### Materials Needed:

* Petri dishes
* LB broth high salt (5 g)
* Bacto agar (5 g)
* Distilled water (200 mL)
* Erlenmeyer flask
* Scale
* Wrap
* Microwave
* Erythromycin (80 µL)

###### Experimental Procedure:

1. Label petri dishes with date, team number, and names.
2. Weigh 5 g LB high salt, 3 g agar, and 200 mL distilled water and pour into the flask.
3. Microwave for 2 min 50 sec, until the solution turns transparent yellow with no solids.
4. Cover the flask opening with wrap and poke a small hole.
5. Pipette 80 µL erythromycin into the solution and swirl gently.
6. Pour ~20 mL into each petri dish (half height).
7. Avoid bubbles since it is hard to distinguish between bacteria colonies and solidified bubbles.
8. Allow agar to solidify at room temperature.

![Pouring LB + agar into petri dishes.](https://static.igem.wiki/teams/5773/experiments/exp-figure4.avif)

Figure 4 (Pouring the LB and Bacto Agar onto the petri dish.)

#### Session Agenda #5: Plating Bacteria

**Experimental Principle:** Spreading bacteria evenly on agar ensures individual cells from colonies. Incubation at optimal temperature promotes growth.

###### Materials Needed:

* Solidified agar plates with erythromycin
* Test tubes with recovered E. coli
* Micropipette + sterile tips
* Sterilized spreader
* Shaking Incubator (37 °C)

###### Experimental Procedure:

1. Pipette 100 µL bacterial culture onto the center of the agar plate on the petri dish.
2. Place a sterilized spreader on the plate; spread bacteria evenly across the surface.
3. Place the plates inside the 37 °C shaking incubator for 24 hours.

![Spreading bacteria with sterilized spreader.](https://static.igem.wiki/teams/5773/experiments/exp-figure5.avif)

Figure 5 (Using the sterilized spreader to evenly distribute the bacteria onto the agar plate.)

#### Session Agenda #6: Transferring Grown Bacteria to Test Tube Solutions

**Experimental Principle:** To extract the bacteria that grew on the agar plates, colonies were transferred into fresh liquid media so the bacteria can multiply, producing more plasmid copies.

### 2nd Wet Lab Session: July 13th, 2025

#### Session Agenda #1: Harvesting Bacterial Cells

**Experimental Principle:** Centrifugation separates bacterial cells from liquid media. The dense cells result at the bottom while the LB medium remains above.

###### Materials Needed:

* 4 bacterial culture tubes
* Refrigerated centrifuge
* Ice bucket

###### Experimental Procedure:

1. Centrifuge the 4 bacterial culture tubes at 3220 rpm, 4 °C, for 3 min.
2. Make sure they are balanced across the rotor.
3. 2. Pour out the LB media into a waste container, leaving only the pelleted bacteria.

![Centrifuging bacterial culture tubes.](https://static.igem.wiki/teams/5773/experiments/exp-figure6.avif)

Figure 6 (Centrifuge the 4 bacterial culture tubes for 3 min at 4 °C, 3220 rpm.)

#### Session Agenda #2: Plasmid Extraction

**Experimental Principle:** The bacterial cells are lysed to release DNA. Potassium acetate neutralizes the solution, precipitating proteins and chromosomal DNA, leaving plasmid only.

###### Materials Needed:

* EX Pure Plasmid Kit
* S1 Resuspension Buffer (NaOH +RNase)
* S2 Lysis Buffer (alkaline solution, NaOH +SDS)
* S3 Neutralization Buffer (potassium acetate)
* Vortex mixer
* Microcentrifuge
* Microtubes

###### Experimental Procedure:

1. Add 250 µL S1 Resuspension Buffer to the bacterial pellet.
2. Vortex each tube until the suspension is uniform with no clumps.
3. Label fresh microtubes and transfer 300 µL of the suspension to each.
4. Add 250 µL S2 Lysis Buffer to each tube and mix gently.
5. Add 350 µL S3 Neutralization Buffer to each tube.
6. Mix it well until the mixture turns white (precipitate forms).
7. Centrifuge the tubes at 13,000 rpm, 21 °C, for 10 min.
8. After centrifugation, a white pellet forms (cell debris, proteins) with a clear supernatant (plasmid DNA).
9. Pipette 750 µL supernatant plasmid DNA into the DNA binding column.
10. Centrifuge at 13,000 rpm, 25 °C, for 1 min.

![Separating supernatant from pellet for plasmid DNA.](https://static.igem.wiki/teams/5773/experiments/exp-figure7.avif)

Figure 7 (Separation of the bacterial cells from the LB media after centrifugation.)

#### Session Agenda #3: Washing and Eluting Plasmid DNA

**Experimental Principle:** Washing buffers remove proteins, salts, and contaminants, leaving plasmid DNA bound to the membrane. Elution releases purified plasmid DNA into a clean tube.

###### Materials Needed:

* DNA binding column
* AW Washing Buffer (ethanol-based)
* ED Elution Buffer
* Microcentrifuge
* Collection tubes

###### Experimental Procedure:

1. Add 700 µL AW Washing Buffer into the DNA binding column.
2. Centrifuge at 13,000 rpm, 25 °C, for 1 min.
3. Remove all the non-plasmid substances.
4. Centrifuge again at 13,000 rpm, 25 °C, for 1 min to remove the Washing Buffer.
5. Remove the “insert” residues to a new test tube.
6. Add 50 µL ED Elution Buffer directly to the membrane
7. Let stand for 1 min at room temperature.
8. Centrifuge at 13,000 rpm, 25 °C, for 1 min.
9. Remove column insert so that the bottom tube now contains purified plasmid DNA.

![Adding AW washing buffer to column.](https://static.igem.wiki/teams/5773/experiments/exp-figure8.avif)

Figure 8 (Adding 700 µL of AW Washing Buffer into the DNA binding column.)

#### Session Agenda #4: DNA Quantification

**Experimental Principle:** Nanodrop uses UV absorbance to measure DNA concentration & purity.

###### Materials Needed:

* Nanodrop spectrophotometer
* ED buffer
* Micropipette + tips
* Alcohol wipes

###### Experimental Procedure:

1. Open Nanodrop software and select dsDNA mode.
2. Test ED buffer for the control.
3. Clean pedestal with alcohol wipes.
4. Load 2 µL of each DNA sample, close the lid, and measure the concentration.

###### Results for DNA Concentration:

| Group | 1st Measure (ng/µL) | 2nd Measure (ng/µL) | 3rd Measure (ng/µL) |
| --- | --- | --- | --- |
| Group 1 | 38.3 | 40.1 | 40.8 |
| Group 2 | 44.0 | 49.7 | 31.5 |
| Group 3 | 56.9 | 47.7 | 46.8 |
| Group 4 | 77.1 | 33.6 | 38.3 |

Results for DNA Concentration.

###### Results for Final DNA Concentration and Purity Ratio:

| Group | ng/µL | A260/A280 | A260/A230 |
| --- | --- | --- | --- |
| Group 1 | 38.3 | 2.01 | 2.39 |
| Group 2 | 44.0 | 1.92 | 1.54 |
| Group 3 | 56.9 | 1.99 | 1.35 |
| Group 4 | 77.1 | 1.88 | 0.86 |

Final DNA Concentration and Purity.

![Loading samples onto the NanoDrop lens.](https://static.igem.wiki/teams/5773/experiments/exp-figure9.avif)

Figure 9 (Loading 2 µL of each sample onto the nanodrop lens.)

### 3rd Wet Lab Session: July 20th, 2025

#### Session Agenda #1: SDS-PAGE Sample Preparation

**Experimental Principle:** Samples of different conditions in the presence of the plasmid and whether Lactobacillus reuteri underwent lysis to assess the production of the pgxC protein.

###### Materials Needed:

* 2 Lactobacillus reuteri-containing solutions, one treated with plasmid and the other not.
* 4 small test tubes
* Centrifuge
* Lysis buffer
* Vortex machine
* Sonicator

###### Experimental Procedure:

1. Centrifuge two 50 mL test tubes, each containing the solution of no-plasmid L. reuteri and plasmid-inserted L. reuteri , for 2 min at 3000rpm and 4 °C.
2. Pipette 5mL of the centrifuged solution, ideally only pulling out the liquid from each centrifuged test tube, and transfer them into small test tubes. Label the small test tubes as “+ plasmid solution,” “- plasmid solution.”
3. Discard the rest of the solution, leaving only the L. reuteri cells in each test tube.
4. Add 10mL of lysis buffer to each of the test tubes containing Lactobacillus reuteri, which functions to burst the cells and to stabilize the pgxC protein.
5. Vortex both test tubes that contain the lysis buffer and Lactobacillus reuteri for approximately 45 seconds to attain a homogeneous solution without clumped cells.
6. Transfer the solution into smaller tubes to fit them into the sonicator.
7. Insert both test tubes into the Sonicator (“Ultrasonic Processor”) to burst the cells.
8. Run the sonicator for 30 seconds, set at pulse 1 sec/1(1 sec on, 1 sec off), amp 1/20%.
9. Repeat for 30 seconds, set at pulse 1 sec/1(1 sec on, 1 sec off), amp 1/20%.
10. Run the sonicator again for 1 minute, set at pulse 1 sec/1, amp 1/20%.
11. Run the sonicator again for 1 minute, set at pulse 1 sec/1, amp 1/40%.
12. Run the sonicator again for 2 minutes, set at pulse 1 sec/1, amp 1/40%.
13. Centrifuge both sonicated test tubes at 3000rpm and 19 °C for 2 minutes to segregate the cell leftovers, which will be submerged after centrifuging, and the pgxC protein.
14. Transfer each solution to test tubes, labelling each as “+ plasmid cell,” “- plasmid cell.”
15. As a result, there are 4 small test tubes labelled as: +plasmid cell, +plasmid solution, -plasmid cell, -plasmid solution.

![Centrifugation step for SDS-PAGE prep.](https://static.igem.wiki/teams/5773/experiments/exp-figure10.avif)

Figure 10 (Centrifuging plasmid-treated and non-treated Lactobacillus reuteri solutions.)

![Adding lysis buffer to cells.](https://static.igem.wiki/teams/5773/experiments/exp-figure11.avif)

Figure 11 (Injecting Lysis buffer into the Lactobacillus reuteri-containing test tube.)

![Vortexing lysates.](https://static.igem.wiki/teams/5773/experiments/exp-figure12.avif)

Figure 12 (Vortexing Lysis buffer and Lactobacillus reuteri cells.)

![Sonication setup.](https://static.igem.wiki/teams/5773/experiments/exp-figure13.avif)

Figure 13 (Sonication of solution containing Lysis buffer and Lactobacillus reuteri.)

###### Session Agenda #2: Gel Electrophoresis

**Experimental Principle:** For the verification of the size of the protein produced, and to confirm that it has the size of the protein desired of production, pgxC.

###### Materials Needed:

* SDS 1x solution
* 4 pgxC samples from previous agenda
* Electrophoresis device
* SDS PAGE Running Buffers
* Protein marker

###### Experimental Procedure:

1. Insert 50 µL SDS 1x solution into all 4 small test tubes and mix the solution in each test tube with the pipette with repeated insert-eject motion.
2. Fill the electrophoresis device (“Mini-PROTEAN Tetra System”) with SDS PAGE Running Buffers loaded on both upper and lower components
3. Load the protein marker 3µL into the first slot of the electrophoresis well.
4. Load the 4 samples, each 10µL, into the electrophoresis well, in the order of: -plasmid solution, +plasmid solution, -plasmid cell, +plasmid cell.
5. The previous step is repeated– now the 4 different samples are each loaded in two wells, resulting in a total of 8 samples loaded.
6. Initiate electrophoresis at 160V, 0.03 Amperes for 40 min.

![Loading SDS-PAGE running buffer.](https://static.igem.wiki/teams/5773/experiments/exp-figure14.avif)

Figure 14 (Loading the SDS PAGE Running Buffer.)

![Loading wells with samples.](https://static.igem.wiki/teams/5773/experiments/exp-figure15.avif)

Figure 15 (Loading the Electrophoresis well.)

###### Session Agenda #3: Gel Staining

**Experimental Principle:** SDG-PAGE gel staining was performed to make the strips of protein that have undergone electrophoresis easily observable.

###### Materials Needed:

* Electrophoresis gel that has undergone SDS-PAGE
* Blue pigmentation solution: Coomassie Brilliant Blue R-250 Staining Solution
* Nutating mixer

###### Experimental Procedure:

1. Remove the Gel after electrophoresis carefully, as it tends to rip easily.
2. Cut off the unnecessary well walls with a green scraper.
3. Submerge the gel into a blue pigmentation solution (Coomassie Brilliant Blue R-250 Staining Solution) for around 1 min, on the nutating mixer.

![Staining gel with Coomassie.](https://static.igem.wiki/teams/5773/experiments/exp-figure16.avif)

Figure 16 (Submerging electrophoresis gel into staining solution.)

### 4th Wet Lab Session: July 27th, 2025

#### Session Agenda #1: Bradford Assay Sample Preparation

**Experimental Principle:** To conduct the Bradford Assay, through which the concentration of pgxC can be verified, the following four samples need to be prepared: +plasmid cell, +plasmid solution, -plasmid cell, -plasmid solution.

###### Materials Needed:

* 2 Lactobacillus reuteri-containing solutions, one treated with plasmid and the other not.
* 4 small test tubes
* Centrifuge
* Lysis buffer
* Vortex machine
* Sonicator

###### Experimental Procedure:

1. Centrifuge two 50 mL test tubes, each containing the solution of no-plasmid Lactobacillus reuteri and plasmid-inserted L. reuteri, for 3 min at 3000 rpm and 4 °C.
2. Pipette 1000 μl of the centrifuged 50 mL test tubes into small sample test tubes, tagging each as plasmid (+) solution and plasmid (-) solution.
3. Discard the centrifuged solution in the 50 mL test tubes, leaving only the clumped cells.
4. Pipette 8.5 mL of lysis buffer into each 50 mL test tube, using the pipette mixing technique for a homogeneous mixture of cells and lysis buffer.
5. Transfer the solutions into smaller test tubes for sonication.
6. Sonication for 15 min at 1/40% amplitude, pulse 3 sec/3.

![Sonication before Bradford assay.](https://static.igem.wiki/teams/5773/experiments/exp-figure17.avif)

Figure 17 (Sonication of solution containing Lysis buffer and Lactobacillus reuteri.)

#### Session Agenda #2: Bradford Assay

**Experimental Principle:** Assessing the concentration of the pgxC protein.

###### Materials Needed:

* 6 small test tubes
* 50 mL test tube
* Distilled water
* Bradford Dye Solution
* 94 well
* Incubator
* Spectrophotometer

###### Experimental Procedure:

1. Label 6 Test tubes from S0 to S5.
2. Pipette as follows:

| Tube # | S0 | S1 | S2 | S3 | S4 | S5 |
| --- | --- | --- | --- | --- | --- | --- |
| Distilled Water (mL) | 20 | 19 | 18 | 17 | 16 | 15 |
| BSA standard solution (mL) | 0 | 1 | 2 | 3 | 4 | 5 |

S0–S5 preparation for Bradford standards.

3. Label a test tube as “Bradford Dye.” Pipette 1440 μl of distilled water into the test tube and 360 μl of Bradford Dye solution.
4. In the 94-well, pipette 195 μl of Bradford Dye solution into 10 different wells.
5. In each of the 10 Bradford Dye solutions, pipette 5 μl each of the 6 solutions of S0 to S5 and 4 solutions of plasmid (+) solution and plasmid (-) solution, and plasmid (+) cell and plasmid (-) cell from the sonicated test tubes from the previous agenda.
6. Incubate the 94-well with lid on for 3 minutes at 37 °C.
7. Determine observance of the 94 well using the Spectramax iD3 spectrophotometer, wavelength at 595nm, set at no shake.

![Preparing S0–S5 tubes.](https://static.igem.wiki/teams/5773/experiments/exp-figure18.avif)

Figure 18 (Preparing the S0-S5 tubes.)

![Placing 94-well plate in spectrophotometer.](https://static.igem.wiki/teams/5773/experiments/exp-figure19.avif)

Figure 19 (Inserting the 94-well plate into the spectrophotometer.)

### 5th Wet Lab Session: August 3rd, 2025

#### Session Agenda #1: DNS Reagent Preparation

**Experimental Principle:** To determine the ability of the pgxC protein to degrade pectin, the DNS assay is utilized, which determines the concentration of sugar upon sugar degradation.

###### Materials Needed:

* 3,5-Dinitrosalicylic acid (DNS)
* Sodium potassium tartrate
* Sodium hydroxide
* Distilled water
* Weighing boats
* Test Tube

###### Experimental Procedure:

1. Label 3 weighing boats each as “DNS,” “SPT,” and “NaOH,” and weigh each component with an electronic scale. Add 0.5g DNS, 0.8g NaOH to the test tube
2. Add 30 mL of distilled water to the same tube, then shake the tube for homogeneity.
3. Add 15 g of sodium potassium tartrate and continue stirring until fully dissolved.
4. Adjust volume to 50 mL with distilled water.

![Weighing DNS.](https://static.igem.wiki/teams/5773/experiments/exp-figure20.avif)

Figure 20 (Weighing the 3,5-Dinitrosalicylic acid (DNS).)

![Adding potassium tartrate.](https://static.igem.wiki/teams/5773/experiments/exp-figure21.avif)

Figure 21 (Adding potassium tartrate.)

###### Session Agenda #2: Preparation of Evaluation Points for the DNS Assay

**Experimental Principle:** To run the DNS assay, there needs to be benchmarking points to draw a standard curve to evaluate the concentration of sugar that occurs when pgxC degrades pectin.

###### Materials Needed:

* D-galacturonic acid
* Distilled water
* 6 Test tubes

###### Experimental Procedure:

1. Dissolve 100 mg of D-galactronic acid in 100 mL of distilled water as a stock.
2. Label 6 test tubes based on concentration:

|  | Tube 1 | Tube 2 | Tube 3 | Tube 4 | Tube 5 | Tube 6 |
| --- | --- | --- | --- | --- | --- | --- |
| Pipette distilled water accordingly to the 6 tubes. | — | | | | | |
| Pipette D-galactronic acid accordingly to the 6 tubes. | — | | | | | |

Preparation of standard solutions.

![Standard solutions for DNS curve.](https://static.igem.wiki/teams/5773/experiments/exp-figure22.avif)

Figure 22 (Preparation of standard solutions)

#### Session Agenda #3: Enzymatic Reaction with Pectin Preparation

**Experimental Principle:** We prepared conditions for pgxC to react with pectin to find the optimal time of incubation for pgxC to most efficiently degrade pectin molecules.

###### Materials Needed:

* 8 Test Tubes
* 1% Pectin Solution in Sodium Acetate Buffer (pH 6.0)
* +/- plasmid solutions
* Sterilization Cabinet
* Alcohol Lamp
* Incubator
* Spectrophotometer
* DNS reagent solution from Agenda 1
* 96-well

###### Experimental Procedure:

1. Label 8 test tubes: 4 plasmid and 4 no plasmid. Each of the four “plasmid” / “no plasmid” is further labelled with “0hr,” “12hr,” “24hr,” and “48hr.”
2. Pipette 4.5mL of 1% pectin solution in sodium acetate buffer (pH 6.0) into all test tubes.
3. Pipette 500 microliters + plasmid solution or - plasmid solution (bacterial supernatant) into the 8 tubes accordingly, referring to the label on each test tube. Perform the pipetting inside the sterilization cabinet with the alcohol lamp.
4. Incubate the test tubes that contain the + plasmid solution and - plasmid solution at 37°C for 0 hours, 12 hours, 24 hours, and 48 hours, referring to the label on test tube.
5. Immediately after each incubation, centrifuge the test tube if needed. Pipette 1 mL of the solution from the test tube into the 96-well, then mix with 500 µL of the DNS reagent solution for the DNS assay. Conduct the DNS assay with a Spectrophotometer.

![Pipetting pectin buffer.](https://static.igem.wiki/teams/5773/experiments/exp-figure23.avif)

Figure 23 (Pasteur pipetting the 1% Pectin Solution in Sodium Acetate Buffer.)

![Pipetting plasmid solutions in cabinet.](https://static.igem.wiki/teams/5773/experiments/exp-figure24.avif)

Figure 24 (Pipetting plasmid solutions in sterilization cabinets.)

### 6th Wet Lab Session: August 10th, 2025

#### Session Agenda #1: Fruit Peel Sample Preparation

**Experimental Principle:** Fruit peels contain pectin (a polysaccharide). Preparing peel extracts creates substrate samples to test enzymatic degradation. Centrifugation separates soluble components from large solid debris.

###### Materials Needed:

* Fruits: Korean pear, watermelon, banana, kiwi, tomato (x4)
* Balance
* Distilled water
* Blender/grinder
* 50 mL conical tubes
* Centrifuge

###### Experimental Procedure:

1. Collect peel from each fruit:

* Watermelon: 78.9 g
* Banana: 53.09 g + 50 mL water
* Kiwi: 29.61 g
* Tomato: 42.63 g
* Pear: 52.62 g

2. Add 50 mL of distilled water to each sample.
3. Grind each fruit peel + water until homogenized into sludge.
4. Transfer each slurry into labeled 50 mL conical tubes.
5. Centrifuge tubes at 2000 rpm, 22 °C, for 2 min.
6. After spinning, collect the liquid phase and avoid pellet debris.

![Peeling watermelon.](https://static.igem.wiki/teams/5773/experiments/exp-figure25.avif)

Figure 25 (Peeling the Watermelon.)

![Tomato peel segregation.](https://static.igem.wiki/teams/5773/experiments/exp-figure26.avif)

Figure 26 (Tomato peel segregation.)

![Ground peel samples.](https://static.igem.wiki/teams/5773/experiments/exp-figure27.avif)

Figure 27 (Grinded Peel Samples.)

![Centrifuging fruit samples.](https://static.igem.wiki/teams/5773/experiments/exp-figure28.avif)

Figure 28 (Centrifuging the fruit samples.)

#### Session Agenda #2: Enzyme Reaction Preparation

**Experimental Principle:** The goal is to test enzyme activity on fruit peel extracts. Adding plasmid solution (+ vs -) allows comparison of enzyme-expressing vs non-expressing samples. DNS assay detects reducing sugars released from polysaccharide breakdown.

###### Materials Needed:

* Fruit peel supernatants
* 10 small test tubes
* Plasmid solution (enzyme-expressing)
* Plasmid solution (control)
* Micropipette + sterile tips
* Vortex mixer
* DNS assay reagent
* Heat block / Dry bath heat
* Additional 10 small test tubes

###### Experimental Procedure:

**Step A: Reaction Setup**

1. Label 10 small test tubes: 2 for each fruit (one “+”, one “-”)
2. Pipette 300 µL fruit peel extract into each tube, avoiding chunks.
3. To “+” tubes, add 200 µL + plasmid solution.
4. To “-” tubes, add 200 µL - plasmid solution.
5. Vortex all tubes for 5 sec.

**Set B: DNS Assay Prep**

1. Label another 10 small test tubes: 2 for each fruit (one “+”, one “-”)
2. Pipette 50 µL DNS solution into each tube.
3. Add 50 µL of each enzyme reaction sample into the corresponding DNS tube.
4. Vortex all DNS tubes to mix.
5. Dry bath heat all DNS tubes at 100 °C for 5 min.
6. DNS reacts with reducing sugars to produce a colored product.

![Vortexing DNS tubes.](https://static.igem.wiki/teams/5773/experiments/exp-figure29.avif)

Figure 29 (Vortexing the DNS tubes.)

![Dry bath heating DNS tubes.](https://static.igem.wiki/teams/5773/experiments/exp-figure30.avif)

Figure 30 (Dry bath heating all DNS tubes.)

#### Session Agenda #3: Absorbance Measurement

**Experimental Principle:** DNS assay turns orange-red when reducing sugars are present. Measuring absorbance at 540 nm quantifies enzymatic breakdown of fruit peel polysaccharides.

###### Material Needed:

* DNS assay reaction tubes (from Agenda 2)
* 96-well microplate
* Micropipette + tips
* Plate reader (set to 540 nm)

###### Experimental Procedure:

1. Label wells on the 96-well plate clearly (fruit name, +/-).
2. Pipette 80 µL of each DNS reaction into its assigned well.
3. Add one well with DNS reagent only (blank/control).
4. Place plate into spectrophotometer/plate reader.
5. Measure absorbance at 540 nm.
6. Record and compare absorbance values (+ vs - plasmid).

![Labeling 96-well plate.](https://static.igem.wiki/teams/5773/experiments/exp-figure31.avif)

Figure 31 (96-well plate labelled.)

![Observation before reading results.](https://static.igem.wiki/teams/5773/experiments/exp-figure32.avif)

Figure 32 (Observer Before Results.)

![Absorbance readout.](https://static.igem.wiki/teams/5773/experiments/exp-figure33.avif)

Figure 33 (Absorbance Numerical Results.)

![Post-reaction color differences.](https://static.igem.wiki/teams/5773/experiments/exp-figure34.avif)

Figure 34 (Observer Results Color.)

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# Results

* [Research Project Goal](#project_goal)
* [Result 1](#result1)
* [Result 2](#result2)
* [Result 3](#result3)
* [Result 4](#result4)
* [Result 5](#result5)
* [Summary of Achievements](#achievements)
* [References](#references)

## Research Project Goal

The goal of our iGEM project was to design and implement a novel microbial system that used Lactobacillus reuteri, a Generally Recognized as Safe (GRAS) probiotic strain, as a chassis for fruit peel degradation. A composite genetic circuit was constructed incorporating the ermB promoter for strong constitutive expression, the Usp45 engineered secretion signal to facilitate extracellular release, and the pgxC gene encoding polygalacturonase, an enzyme capable of cleaving pectin polymers abundant in fruit peels. The plasmid backbone pTRKH3 was used for stable replication and expression in L. reuteri. Through this strategy, we aimed to achieve efficient secretion of active polygalacturonase into the extracellular environment, enabling direct breakdown of fruit peel pectin into galacturonic acid and simpler sugars. By coupling probiotic safety with enzymatic efficiency, this system addressed the global issue of fruit waste accumulation while contributing to sustainable bioconversion. Furthermore, the project envisioned downstream applications, such as using the resulting degraded biomass as a substrate for bio-based products (biofuels, animal feed, or prebiotic supplements), thereby establishing a circular and eco-friendly waste-to-resource platform.

### Result 1: Synthesizing pgxC with Usp45 Secretion Peptide Sequence

![Proposed engineered pgxC protein sequence fused with the USP45 secretion signal and cloned into the pTRKH3 backbone vector](https://static.igem.wiki/teams/5773/results/figure-1.webp)

Figure 1. Proposed engineered pgxC protein sequence fused with the USP45 secretion signal and cloned into the pTRKH3 backbone vector.

The wild-type pgxC protein sequence (highlighted in red) was compared with the secretion signal sequence derived from USP45 (highlighted in blue). To facilitate secretion of the engineered enzyme, the N-terminal region of pgxC was fused in-frame with the USP45 signal peptide, generating the proposed engineered pgxC construct (bottom panel). This design ensures that the protein maintains its catalytic core while acquiring an additional leader sequence that directs its secretion outside of the bacterial host cell. After obtaining the pTRKH3 plasmid backbone vector, the engineered pgxC with USP45 secretion signal was synthesized and cloned using standard restriction enzyme–based cloning methods (Figure 1).

The synthesis process involved codon optimization for efficient bacterial expression and seamless junction design between the USP45 signal peptide and pgxC coding sequence. Restriction sites were strategically introduced to allow precise ligation of the engineered insert into the pTRKH3 multiple cloning site without disrupting the reading frame. Following successful assembly, the construct was verified by sequencing, ensuring correct orientation and fidelity of the engineered sequence. The final recombinant plasmid, pTRKH3-USP45-pgxC, was designed to enable secretion of polygalacturonase enzymes into the extracellular medium, thus improving substrate accessibility and overall enzyme efficiency (Figure 2). Detailed steps of plasmid construction and cloning strategy are described in the engineering section.

![ The plasmid vector map of pTRKH3-pgxC_modified signal peptide constructed for this study.](https://static.igem.wiki/teams/5773/results/figure-2.webp)

Figure 2. The plasmid vector map of pTRKH3-pgxC\_modified signal peptide constructed for this study.

The recombinant plasmid is 9,585 bp in size and was designed to enable secretion and expression of the engineered pgxC enzyme in a bacterial host system. The modified construct incorporates the USP45 signal peptide fused to the N-terminus of the pgxC coding sequence, driven by the strong ermB promoter to ensure robust transcription. The plasmid backbone, derived from pTRKH3, provides stability and compatibility for Gram-positive bacterial expression systems. Multiple cloning sites (MCS) are available and flanked by numerous restriction enzyme recognition sequences, which allow flexible cloning and potential future modifications.

In addition to the engineered pgxC region, the plasmid carries an antibiotic resistance marker for selective growth, a replication origin for stable maintenance, and regulatory elements such as the tet promoters and cat promoter, which provide tunable control over gene expression. The detailed arrangement of restriction sites around the insert ensures that the engineered signal peptide and coding sequence were seamlessly integrated without disrupting the reading frame.

### Result 2: Transformation of pTRKH3-pgxC-Modified Signal Peptide in Both E.coli and L. reuteri

![Representation of agar plates after transformation of the pTRKH3-pgxC plasmid in L. reuteri (electroporation)](https://static.igem.wiki/teams/5773/results/figure-3.webp)

Figure 3. Representation of agar plates after transformation of the pTRKH3-pgxC plasmid in L. reuteri (electroporation).

The agar plate images illustrate the outcomes of transformation with the engineered pTRKH3-pgxC plasmid (Figure 3). The negative control plate (left, without plasmid) showed no colony growth, confirming the absence of background resistance. In contrast, the experimental plate (right, with plasmid) displayed abundant colony formation, indicating successful uptake and expression of the recombinant construct by L. reuteri competent cells. The observed colonies arose from bacteria harboring the pTRKH3-pgxC plasmid, which carries an antibiotic resistance marker, thereby allowing selective growth.

This result confirms the efficient introduction of the engineered plasmid into both E. coli and L. reuteri. Transformation of E. coli, used primarily as a cloning host, was achieved via the heat shock method, whereas L. reuteri, the intended expression host, was successfully transformed using electroporation. The ability to establish the construct in both bacterial systems demonstrates its broad host compatibility. The high density of colonies observed on the plasmid-containing plates highlights robust transformation efficiency, providing a solid foundation for downstream applications such as plasmid propagation, protein secretion analysis, and functional assays of the engineered pgxC enzyme.

### Result 3: Detection of Secreted pgxC Protein in Plasmid-Transformed L. reuteri

![ Detection of secreted pgxC protein in Lactobacillus reuteri. (Left) Illustration showing separation of cell pellet and supernatant, with pgxC proteins detected in the extracellular fraction. (Right) SDS-PAGE gel analysis of culture supernatant (S) and cell lysate (L) from L. reuteri strains with and without plasmid expression. A distinct protein band corresponding to ~48.5 kDa, consistent with the predicted size of pgxC, was observed in the supernatant and lysate of cells harboring the pgxC plasmid, but not in controls lacking the plasmid.](https://static.igem.wiki/teams/5773/results/figure-4.webp)

Figure 4. Detection of secreted pgxC protein in Lactobacillus reuteri. (Left) Illustration showing separation of cell pellet and supernatant, with pgxC proteins detected in the extracellular fraction. (Right) SDS-PAGE gel analysis of culture supernatant (S) and cell lysate (L) from L. reuteri strains with and without plasmid expression. A distinct protein band corresponding to ~48.5 kDa, consistent with the predicted size of pgxC, was observed in the supernatant and lysate of cells harboring the pgxC plasmid, but not in controls lacking the plasmid.

To confirm expression and secretion of pgxC, proteins from both the culture supernatant (S) and cell lysate (L) were analyzed by SDS-PAGE (Figure 4). A distinct band corresponding to approximately 48.5 kDa, which matched the predicted molecular weight of pgxC, was observed in both the supernatant and cell lysate of L. reuteri containing the pgxC plasmid. In contrast, no such band was detected in the negative control samples lacking the plasmid. The presence of the pgxC band in the supernatant indicated that the engineered Usp45 secretion signal successfully directed protein export outside the cell. These results demonstrated that L. reuteri carrying the recombinant plasmid not only expressed pgxC but also secreted it into the extracellular medium, validating the effectiveness of the designed secretion system.

The successful detection of pgxC in the extracellular fraction demonstrated that our engineered secretion system was functional, enabling L. reuteri to release the enzyme directly into the surrounding medium. This is a critical outcome for the project, as extracellular secretion eliminates the need for costly cell disruption or purification steps and allows the enzyme to act directly on fruit peel biomass. Since pectin is a major structural component of fruit peels, the secretion of active pgxC provides a practical foundation for efficient biodegradation of agricultural waste. Furthermore, the use of a probiotic chassis such as L. reuteri enhances the safety and scalability of this system, opening applications in sustainable waste management.

### Result 4: Quantifying pgxC Secreted Proteins Using BSA Assay

![ Quantification of secreted proteins using the Bradford assay. (Top) BSA standard curve generated by measuring absorbance at 595 nm across a concentration range of 0–0.5 µg/µL. The linear regression equation (y = 0.401x + 0.091, R² = 0.94) was used to calculate protein concentrations in experimental samples. (Bottom) Protein concentrations in the culture supernatants of Lactobacillus reuteri with and without the pgxC plasmid. Samples with the plasmid showed a significantly higher protein concentration (0.10 ± 0.01 µg/µL) compared to the control without plasmid (0.04 ± 0.005 µg/µL), confirming enhanced extracellular protein expression.](https://static.igem.wiki/teams/5773/results/figure-5.webp)

Figure 5. Quantification of secreted proteins using the Bradford assay. (Top) BSA standard curve generated by measuring absorbance at 595 nm across a concentration range of 0–0.5 µg/µL. The linear regression equation (y = 0.401x + 0.091, R² = 0.94) was used to calculate protein concentrations in experimental samples. (Bottom) Protein concentrations in the culture supernatants of Lactobacillus reuteri with and without the pgxC plasmid. Samples with the plasmid showed a significantly higher protein concentration (0.10 ± 0.01 µg/µL) compared to the control without plasmid (0.04 ± 0.005 µg/µL), confirming enhanced extracellular protein expression.

A standard curve was generated using BSA concentrations ranging from 0 to 0.5 µg/µL, and absorbance at 595 nm was measured after Bradford reagent addition (Figure 5). The resulting linear regression equation was y = 0.401x + 0.091 with an R² value of 0.94, indicating a strong correlation between absorbance and protein concentration. Using this equation, protein concentrations were quantified in the culture supernatants of Lactobacillus reuteri. Samples without plasmid showed a protein concentration of approximately 0.04 µg/µL, while samples containing the pgxC plasmid exhibited a higher concentration of 0.10 µg/µL, suggesting successful expression and secretion of recombinant proteins.

The increase in protein concentration in the supernatant of plasmid-containing L. reuteri compared with the control confirmed that recombinant pgxC expression contributed to extracellular protein content. Although the total protein concentration measured included both secreted pgxC and native proteins, the relative increase in the plasmid-bearing condition is consistent with the SDS-PAGE results showing a clear 48.5 kDa pgxC band in the supernatant.

The strong linearity of the BSA standard curve (R² = 0.94) validated the accuracy of the Bradford assay in this experiment, though minor deviations from an R² of 1 likely reflect pipetting variability or uneven dye–protein binding efficiency. Together, these findings demonstrated that the engineered secretion system enabled detectable increases in extracellular protein concentration, supporting the functionality of pgxC expression and secretion in L. reuteri.

### Result 5: Quantifying D-galacturonic Acid after Incubating with Pectin and pgxC Secreted Proteins Using DNS Assay

![ Standard curve for D-galacturonic acid quantification using the DNS assay. The standard curve was generated by measuring absorbance at 540 nm for increasing concentrations of D-galacturonic acid (0–1.0 mg/mL). The linear regression equation (A = 1.207·C + 0.016) with an R² value of 1.000 indicated excellent linearity across the tested range. This curve was subsequently used to calculate the concentration of reducing sugars released after incubation of pectin with pgxC-secreted proteins from Lactobacillus reuteri.](https://static.igem.wiki/teams/5773/results/figure-6.webp)

Figure 6. Standard curve for D-galacturonic acid quantification using the DNS assay. The standard curve was generated by measuring absorbance at 540 nm for increasing concentrations of D-galacturonic acid (0–1.0 mg/mL). The linear regression equation (A = 1.207·C + 0.016) with an R² value of 1.000 indicated excellent linearity across the tested range. This curve was subsequently used to calculate the concentration of reducing sugars released after incubation of pectin with pgxC-secreted proteins from Lactobacillus reuteri.

D-galacturonic acid was chosen as the calibration standard because it represents the major monosaccharide building block of pectin, a complex polysaccharide abundant in fruit peels. Pectin is composed mainly of α-(1→4)-linked D-galacturonic acid residues, often methyl-esterified or acetylated, forming homogalacturonan regions and rhamnogalacturonan domains. During enzymatic degradation, polygalacturonases such as pgxC hydrolyze the glycosidic linkages between these galacturonic acid units. This cleavage releases free D-galacturonic acid or shorter oligogalacturonides, which can then be detected as reducing sugars using the DNS assay. Thus, using D-galacturonic acid as the standard ensures that the assay directly reflects the extent of pectin depolymerization catalyzed by pgxC, providing an accurate measure of enzymatic activity during fruit peel degradation.

To evaluate the enzymatic activity of secreted pgxC, the DNS assay was performed to quantify the release of reducing sugars, specifically D-galacturonic acid, after incubating fruit peel pectin with culture supernatants. A standard curve generated using pure D-galacturonic acid (0–1.0 mg/mL) showed a strong linear correlation between concentration and absorbance at 540 nm (A = 1.207·C + 0.016, R² = 1.000) (Figure 6).

![ Quantification of D-galacturonic acid after 0, 12, 24, 48h incubation of supernatant produced from control (no pgxC) and engineered (pgxC) samples. Values are shown as mean ± SD from three replicates.](https://static.igem.wiki/teams/5773/results/figure-7.webp)

Figure 7. Quantification of D-galacturonic acid after 0, 12, 24, 48h incubation of supernatant produced from control (no pgxC) and engineered (pgxC) samples. Values are shown as mean ± SD from three replicates.

To further assess the kinetics of pectin degradation, supernatants from control (no pgxC) and engineered (pgxC-expressing) Lactobacillus reuteri were incubated with pectin for 0, 12, 24, and 48 hours, and the release of reducing sugars was quantified by DNS assay (Figure 7). In the engineered strain, absorbance values at 540 nm increased steadily over time, reaching 0.35 at 12 h, 0.61 at 24 h, and 0.67 at 48 h, reflecting continuous production of D-galacturonic acid. By contrast, the control strain showed only minimal changes in absorbance throughout the incubation period, remaining below 0.1 even at 48 h. These results demonstrated that the pgxC-expressing strain actively degraded pectin in a time-dependent manner, while the control strain exhibited negligible enzymatic activity. Together, these findings confirm that secreted pgxC enabled effective extracellular breakdown of fruit peel pectin, validating the engineered system’s functional role in waste bioconversion.

![Comparison of D-galacturonic acid release from fruit peels after 24 h incubation with control and engineered Lactobacillus reuteri supernatants. DNS assay was performed to quantify reducing sugars at 540 nm in extracts of banana, kiwi, pear, tomato, and watermelon peels. Orange bars represent control cultures (no pgxC), and blue bars represent engineered strains expressing pgxC. Values are shown as mean ± SD from three replicates.](https://static.igem.wiki/teams/5773/results/figure-8.webp)

Figure 8. Comparison of D-galacturonic acid release from fruit peels after 24 h incubation with control and engineered Lactobacillus reuteri supernatants. DNS assay was performed to quantify reducing sugars at 540 nm in extracts of banana, kiwi, pear, tomato, and watermelon peels. Orange bars represent control cultures (no pgxC), and blue bars represent engineered strains expressing pgxC. Values are shown as mean ± SD from three replicates.

To evaluate real-world applicability, we tested fruit peels from banana, kiwi, pear, tomato, and watermelon as substrates (Figure 8). After 24 h incubation, engineered L. reuteri supernatants produced significantly higher levels of D-galacturonic acid compared to controls, reflecting efficient peel degradation. Pear and tomato peels exhibited the greatest release (~0.42–0.51 mg/mL), followed by kiwi (~0.30 mg/mL) and banana (~0.22 mg/mL). Watermelon peels produced minimal sugars (<0.1 mg/mL), consistent with their lower pectin content.

These results demonstrate both the substrate range and specificity of the engineered strain, highlighting its suitability for valorizing citrus- and pear-rich agricultural waste streams.

### Summary of Achievements

Through iterative design, verification, and testing, we successfully engineered *L. reuteri* to:

* Express codon-optimized *pgxC* fused with a secretion peptide.
* Stably maintain the recombinant construct and efficiently transform into *L. reuteri*.
* Secrete enzymatically functional *pgxC* extracellularly, confirmed by band detection and protein quantification.
* Hydrolyze fruit peel pectin into detectable levels of galacturonic acid, validated across multiple fruit substrates.

Together, these results establish our system as a practical proof-of-concept for fruit peel bioconversion. By combining probiotic safety with efficient extracellular enzyme action, this platform lays the groundwork for waste valorization routes such as feedstock generation, biofuel precursors, and prebiotic supplement production—contributing to a circular and sustainable bioeconomy.

### References

1. de Vos, W. M., & Kleerebezem, M. (2004). Engineering metabolic highways in *Lactobacillus* for dairy and probiotic applications. *Current Opinion in Biotechnology*, 15(5), 486–494. <https://doi.org/10.1016/j.copbio.2004.08.002>.
2. O’Sullivan, D. J., & Klaenhammer, T. R. (1993). High- and low-copy-number *Lactococcus* shuttle cloning vectors with features for clone screening. *Gene*, 137(2), 227–231. [https://doi.org/10.1016/0378-1119(93)90011-Q](https://doi.org/10.1016/0378-1119%2893%2990011-Q).
3. van Asseldonk, M., Rutten, G., Oteman, M., Siezen, R. J., de Vos, W. M., & Simons, G. (1990). Cloning, expression, and nucleotide sequence of *usp45*, a gene encoding a secreted protein from *Lactococcus lactis*. *Journal of Bacteriology*, 172(11), 7126–7136. <https://doi.org/10.1128/jb.172.11.7126-7136.1990>.
4. Jayani, R. S., Saxena, S., & Gupta, R. (2005). Microbial pectinolytic enzymes: A review. *Process Biochemistry*, 40(9), 2931–2944. <https://doi.org/10.1016/j.procbio.2005.03.026>.
5. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. *Analytical Chemistry*, 31(3), 426–428. <https://doi.org/10.1021/ac60147a030>.

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