iGEM 2024 — Team korea-hs

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

Notebook

# Team Korea\_HS Meetings

Date: 03/17/2024

Event: 1st Whole Team Meeting (Virtual)

Agenda: The team was introduced to its members for the first time. Additionally, Professor Lee introduced the team to Synechocystis sp. and the general process involved in the production of bioplastics.

Date: 03/24/2024

Event: 2nd Whole Team Meeting (Virtual)

Agenda: The team was introduced to the team's organization systems, such as the shared Google Drive folder, team group chat, and Instagram. In addition, iGEM registration was explained, along with the leader election process and medal/award criteria.

Date: 03/31/2024

Event: 3rd Whole Team Meeting (Virtual)

Agenda: The team discussed plans for in-person activities and meetings during the summer and received a lecture by Professor Lee on the science behind the production of bioplastic through Synechocystis sp.

Date: 04/07/2024

Event: 4th Whole Team Meeting (Virtual)

Agenda: Candidates for the team leader, co-leader, and sub-team leaders gave their election speeches, which were then followed up by a voting process.

Date: 04/14/2024

Event: 5th Whole Team Meeting (Virtual)

Agenda: The team reviewed award strategies and the various rubrics for special awards. Afterward, Professor Lee delivered a lecture about bacterial culture.

Date: 04/21/2024

Event: 6th Whole Team Meeting (Virtual)

Agenda: The team reviewed the internal and external competition calendar, the 2023 team’s website, criteria and rubrics, and consent forms, and members officially joined the team on the iGEM website.

Date: 04/28/2024

Event: 7th Whole Team Meeting (Virtual)

Agenda: The team conducted a thorough progress check on sub-teams and discussed upcoming safety forms and description deadlines. Professor Lee addressed the role of evolutionary engineering in the team’s project.

Date: 05/19/2024

Event: 8th Whole Team Meeting (Virtual)

Agenda: Professor Lee further explained the role of evolutionary engineering in the team’s project.

Date: 05/26/2024

Event: 9th Whole Team Meeting (Virtual)

Agenda: The team brainstormed characteristics of a good iGEM wiki by examining examples from other teams from previous years. After reviewing a short assessment that tested the members’ understanding of the competition’s judging criteria, the team reviewed upcoming deadlines.

Date: 06/09/2024

Event: 10th Whole Team Meeting (Virtual)

Agenda: The team received a lecture by Professor Lee on Nile staining and fluorescent microscopy to prepare for the upcoming wet lab sessions.

Date: 06/23/2024

Event: 11th Whole Team Meeting (Virtual)

Agenda: The team conducted a thorough progress check on sub-teams and discussed the upcoming deadlines for the Project Promotion Video, specifically focusing on the scriptwriting, and general website theme.

Date: 06/30/2024

Event: 12th Whole Team Meeting (Recording)

Agenda: After briefly reviewing the planned summer activities, the team received a lecture on the detailed steps to note-taking and recording the wet lab experiments in a clear and comprehensive manner for future Wiki write-ups.

Date: 07/06/2024

Event: 13th Whole Team Meeting (In-Person)

Agenda: The team reviewed changes to the 2023/2024 competition, took group photos for the wiki, discussed award strategies, and conducted various in-person surveys at different locations in Seoul, Korea.

Date: 07/21/2024

Event: 14th Whole Team Meeting (Virtual)

Agenda: The team conducted a thorough progress check on sub-teams and discussed award strategies regarding how to document our activities in a coherent and comprehensive manner. In relation to this, the team also went over examples of previous iGEM teams, discussing their strengths and weaknesses regarding our own progress.

Date: 08/03/2024

Event: 15th Whole Team Meeting (Virtual)

Agenda: The team conducted a thorough progress check on sub-teams and received a brief presentation on how the Wiki documentation process works, including its proper format. It was emphasized to the members that this process, along with its finer details, is crucial in shaping the team’s overall first impression.

Date: 08/17/2024

Event: 16th Whole Team Meeting (Virtual)

Agenda: The team conducted a thorough progress check on sub-teams. Each sub-team leader was reminded of how to manage their team’s time effectively to maximize productivity and not fall behind.

Date: 09/08/2024

Event: 17th Whole Team Meeting (Virtual)

Agenda: The team conducted a thorough progress check on sub-teams and went through a list of the approaching deadlines. The members also received a short presentation lecture on the village our team was in, looking over iGEM teams who were in this village last year to compare and contrast our own project with theirs.

Date: 09/15/2024

Event: 18th Whole Team Meeting (Virtual)

Agenda: The team conducted a thorough progress check on sub-teams and received a short presentation lecture on the team’s Grand Jamboree schedule, providing examples and comparing it to last year’s. The members were able to familiarize themselves with what to expect from this year’s schedule.

Date: 09/29/2024

Event: 19th Whole Team Meeting (Virtual)

Agenda: The team conducted a thorough progress check on sub-teams and went through a list of the approaching deadlines. The members were reminded to stay focused and on track, as we were now in the final stages of preparing for iGEM.

# Education & Communication Meetings

Date: 08/31/2024

Event: iGEM TED Talk Summit

Agenda: From 11 PM to 12:30 AM KST, the different club representatives from various schools partook in a group discussion with specific questions related to the SDGs and climate change. Representatives shared what their club or organization does and its relation to the SDGs, spreading awareness and getting to know each other better. Lastly, they were divided into two groups: one creating a business proposal about bioplastics and another building a proposed resolution about the achievement of SDG Goal #17: Partnership for the Goals in relation to SDG Goal #13: Climate Action.

Date: 09/04/2024

Event: Communication with the ASIJ iGEM team

Agenda: Both teams shared their overall projects and discussed the different ways they utilize Synechocystis sp. in their respective projects.

# Interviews & Outreach Meetings

Date: 06/13/2024

Interviewee: Julie Andersen, CEO and Founder of Plastic Oceans [Business Owner]

Agenda: The team asked questions about plastic pollution advocacy and sought advice on spreading awareness on such issues.

Date: 07/21/2024

Interviewee: Haneul Kim, Upcycling Designer [Consumers/Additional]

Agenda: The team visited his studio in Seoul, Korea, to ask questions about his work and his process of upcycling plastic.

Date: 07/21/2024

Interviewee: Seoul Energy Dream Center Representative [Company Employees]

Agenda: The team visited the Seoul Energy Dream Center to learn about green energy and ask its representative questions regarding their pursuits of eco-friendly initiatives.

Date: 07/25/2024

Interviewee: Choroc Maeul Representative [Company Employee]

Agenda: The team interviewed a Choroc Maeul representative to learn about the limitations of bioplastic use in the large-scale packaging of food.

Date: 07/25/2024

Interviewee: YunSeong Im, 247 Pack Representative [Business Owner]

Agenda: The team inquired about the plastic packaging company’s take on bioplastics.

Date: 07/25/2024

Interviewee: Jeong Sung-Il, CEO of Earthform [Business Owner]

Agenda: The team interviewed about Earthform’s production of biodegradable styrofoam and sought advice on the accessibility of PHB bioplastic.

Date: 07/25/2024

Interviewee: Gwangjang Market Juice Vendor [Merchant at Gwang Jang Market]

Agenda: The team asked questions regarding the vendor’s plastic use and awareness of plastic pollution issues.

Date: 07/25/2024

Interviewee: Gwangjang Market Coffee N Waffle Store Owner [Merchant at Gwang Jang Market]

Agenda: The team asked questions regarding the store’s plastic use and awareness of plastic pollution issues.

Date: 07/25/2024

Interviewee: DanDan Box Representatives [Company Employee]

Agenda: The team asked questions regarding the company’s business idea of eco-friendly paper packaging for agricultural products.

Date: 07/25/2024

Interviewee: Vytrus Biotech Representative [Company Employee]

Agenda: The team asked questions regarding the company’s use of plant-based ingredients in plant biotechnology.

Date: 07/25/2024

Interviewee: Plantus Industry Representative [Company Employee]

Agenda: The team asked questions regarding the company’s emphasis on the integration of sustainable technology and innovative development practices.

Date: 07/25/2024

Interviewee: Econnect Representative [Company Employee]

Agenda: The team asked questions regarding the company’s business idea of various eco-friendly plastic alternatives and sought to gain insight into the current market perception of bioplastic adoption.

Date: 07/25/2024

Interviewee: Gwangjang Market Guang Seong Bedding Store Owner [Merchant at Gwang Jang Market]

Agenda: The team asked questions regarding the use of plastic material for the preservation and packaging of various bedding materials sold at the market.

Date: 08/11/2024

Interviewee: Axton Salim, Executive Director of Salim Group [Business Owner]

Agenda: The team asked questions regarding the possible practical limitations of the conversion to bioplastics for large-scale food manufacturers.

# Human Practices Meetings

Date: 03/24/2024

Event: 1st HP Meeting

Agenda: The team got to know each other better and was introduced to the general idea of human practices.

Date: 03/31/2024

Event: 2nd HP Meeting

Agenda: The team completed a brief orientation on the roles and responsibilities of the human practices subgroup.

Date: 04/07/2024

Event: 3rd HP Meeting

Agenda: As a team, we elected the HP leader.

Date: 04/14/2024

Event: 4th HP Meeting

Agenda: The team brainstormed and listed general stakeholders that could be applicable to our project, categorizing them into 4 distinct groups.

Date: 04/21/2024

Event: 5th HP Meeting

Agenda: The team reviewed the guidelines used to source specific stakeholders, discussed the protocols for documenting all the relevant contact information, assigned specific roles for sourcing individual stakeholders, and began the research process.

Date: 04/28/2024

Event: 6th HP Meeting

Agenda: The team completed a progress review of stakeholder sourcing and was briefed on the guidelines for the upcoming outreach process.

Date: 05/19/2024

Event: 7th HP Meeting

Agenda: Individual feedback was provided on each stakeholder sourcing, and the team conducted a thorough quality check. Then, the team made edits and refinements to enhance the sourcing.

Date: 05/26/2024

Event: 8th HP Meeting

Agenda: The team was given a final round of feedback, and time was allocated for making final revisions. The team also reviewed the outreach process and started drafting emails and preparing phone call scripts.

Date: 06/09/2024

Event: 9th HP Meeting

Agenda: The team collaboratively brainstormed a range of relevant interview questions to ask stakeholders in upcoming interviews.

Date: 06/23/2024

Event: 10th HP Meeting

Agenda: The team completed a progress review of the outreach updates and discussed plans for in-person activities.

Date: 07/30/2024

Event: 11th HP Meeting

Agenda: The team completed a progress review of any additional outreach updates and further discussed plans for in-person activities, finalizing the details and questions to ask during stakeholder surveys and interviews.

Date: 07/06/2024

Event: 12th HP Meeting

Agenda: The team conducted sticker surveys and interviews with the general public in the Apgujeong area of Seoul.

Date: 07/18/2024

Event: 1st Collective In-Person Stakeholder Interviews

Agenda: The team visited the Seoul Energy Dream Center to conduct interviews with key stakeholders and also toured Designer Kim Haneul’s studio followed by an interview.

Date: 07/20/2024

Event: 13th HP Meeting

Agenda: The team organized all the interviews conducted so far, distinguished between human practices and integrated human practices, obtained member feedback on interview quality, and discussed the wiki outline specifically for human practices.

Date: 07/25/2024

Event: 2nd Collective In-Person Stakeholder Interviews

Agenda: The team conducted interviews with everyday producers and consumers at the InterCharm (beauty and cosmetics) exhibition and the food and agriculture exhibition, completing over ten interviews at each event.

Date: 08/03/2024

Event: 14th HP Meeting

Agenda: The team reviewed the concept of integrated human practices and categorized stakeholders from all interviews conducted to date into 4 distinct categories.

Date: 08/17/2024

Event: 15th HP Meeting

Agenda: With the interview notes written beforehand, the team began drafting the wiki write-up for human practices after reviewing the appropriate format for the interview reflection section. The team also created a stakeholder interview flowchart as a visual representation.

Date: 08/31/2024

Event: 16th HP Meeting

Agenda: The team reviewed task completion and conducted peer reviews of the interview reflections.

Date: 09/01/2024

Event: 17th HP Meeting

Agenda: The team leader, co-leaders, and HP leader met for an overall progress review and discussed the appropriate next steps.

Date: 09/14/2024

Event: 18th HP Meeting

Agenda: The team reviewed task completion and started discussing how to approach the Integrated HP page. Team members were provided with resources and examples from previous iGEM teams and had the opportunity to familiarize themselves. The team brainstormed and organized how the Wiki page should be structured, formatting the content in the most coherent and logical manner possible.

Date: 09/28/2024

Event: 19th HP Meeting

Agenda: After reviewing task completion, the members were given time to continue with the Wiki documentation process, writing up the page we were responsible for.

# SDG Meetings

Date: 04/09/2024

Event: 1st SDG Meeting

Agenda: The team chose SDG goals to focus on and brainstormed ideas for SDG activities.

Date: 04/16/2024

Event: 2nd SDG Meeting

Agenda: The team added up the SDG activities to commit and planned for the main projects.

Date: 04/23/2024

Event: 3rd SDG Meeting

Agenda: The team went through the roadmaps and the medal criteria.

Date: 05/19/2024

Event: 4th SDG Meeting

Agenda: The team created questions for the online SDG awareness survey and wrote down some interview questions that could be asked or any topics we should get to know about. Also, we individually planned what kind of Instagram posts we would be creating and officially started the Instagram project.

Date: 05/26/2024

Event: 5th SDG Meeting

Agenda: The team went through the homework of sharing our own interview questions that each of us created and filling out the When2meet meeting time. We also went through the Instagram Project of how it is working and the progress in general. Lastly, we finalized the Google survey that we were working on last meeting, and then the leader assigned the task of sharing the survey with at least 3 people to spread awareness and receive responses.

Date: 06/01/2024

Event: 6th SDG Meeting

Agenda: The team went through the homework of sharing the survey with 3 people and was officially introduced to the pamphlet and divided it into 3 sections with each member responsible for a part (about our project, how our project impacts sustainability, the seriousness of the focused issues).

Date: 06/08/2024

Event: 7th SDG Meeting

Agenda: The team checked the homework with the continuation of the Instagram projects, the interview questions for Julie Andersen, and the pamphlet. Then, the team discussed creating a hashtag event and brainstormed ideas.

Date: 06/18/2024

Event: 8th SDG Meeting

Agenda: The team checked the homework with the continuation of Instagram projects, the pamphlet, and the hashtag ideas for the hashtag event. Also, there was an update about the interview with Julie Andersen as it was done before the meeting. Lastly, the team slowly started planning the SDG conference, setting up the agenda and main aim/topic of it.

Date: 06/29/2024

Event: 9th SDG Meeting

Agenda: The team checked through the individual Instagram projects and whether the pamphlet was fully completed. The leader talked about how the in-person general meeting will work, focusing on finishing the individual Instagram project creation, cutting the pamphlets out, doing the in-person survey with the pamphlet distribution, and building ideas with the Children Center education and DIY Bioplastic Creation.

Date: 07/20/2024

Event: 10th SDG Meeting

Agenda: The team shared ideas on what to do in terms of children’s center education, had a short discussion about such ideas with the DIY Bioplastic Kit, and continued planning the SDG Conference.

Date: 07/27/2024

Event: 11th SDG Meeting

Agenda: The team checked the homework with the SDG Conference Plan (adding more ideas) and finalized deciding activities for the Children Center education and dividing roles between the members. Then, the team discussed ideas on how we can expand Instagram and spread more awareness to others.

Date: 08/03/2024

Event: 12th SDG Meeting

Agenda: The team went through the homework check with building ideas for the SDG Conference, finishing the creation for Children Center Education, and sharing interactive Instagram activities. As a team, we decided on a date for the SDG Conference and the leader officially introduced the SDG Wiki's first draft. The team started working on it and divided the roles.

Date: 08/10/2024

Event: 13th SDG Meeting

Agenda: The team went through the homework with building up the Instagram interactive activity and the children’s center education activity. The team also had a mini-check-up session with the Wiki first draft, checking the progress of everyone’s work. Lastly, there was a discussion session about the SDG Conference, finalizing an organized structure, and creating a team poster.

Date: 08/17/2024

Event: 14th SDG Meeting

Agenda: The team had the official Wiki First Draft Mid-checkup and the last, final SDG Conference buildup. The leader also explained how the Bioplastic kit activity would work.

Date: 08/31/2024

Event: SDG Conference

Agenda: The team started the SDG Conference/Ted Talk Summit at 11 PM KST and ended at 12:30 AM KST. The business proposal and the resolution were worked on.

Date: 09/14/2024

Event: 15th SDG Meeting

Agenda: The team checked the homework and started distributing parts of the SDG page write-up to individual members. During the meeting, the team wrote out brief outlines of what content each section should include. By the next meeting, a refined first draft should be ready for feedback and editing.

Date: 09/21/2024

Event: 16th SDG Meeting

Agenda: The team checked the homework and quickly moved over to sharing each individual member’s progress on their portion of the SDG page. During this process, all team members provided constructive feedback to one another, working to improve the content. Afterward, the team brainstormed potential ideas for the official team Instagram page. Ideas ranged from posts to spread awareness to public trivia and challenges.

Date: 09/28/2024

Event: 17th SDG Meeting

Agenda: After checking the homework from our last meeting, the team shared each of their ideas on the Instagram posts. Once everyone had a chance to present their brainstormed ideas, the members voted their top choices. Because a reel to spread awareness regarding plastic pollution was voted on, a few of the other members took on the task of making a card news post to promote and accompany the reel.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

Engineering

PHB Production Through Synechocystis sp.

# Design

# Engineering Synechocystis sp. for Enhanced PHB Production under High pH Conditions

Our engineering strategy aimed to enhance polyhydroxybutyrate (PHB) production in Synechocystis sp. PCC 6803 under high pH culture conditions. This was achieved through an iterative approach involving **Adaptive Laboratory Evolution (ALE)**, high-throughput **PHB production screening**, and **RNA sequencing (RNA-seq)** for transcriptional profiling.

## 1. Adaptive Laboratory Evolution (ALE) for High pH Tolerance

ALE was implemented to evolve Synechocystis towards greater tolerance of alkaline conditions. The experimental design began by gradually increasing the pH from 7.5 to a target of 11.5 in small increments of 0.1 pH units per cycle. Each pH adjustment was followed by a stabilization period, allowing the culture to adapt to the higher pH while maintaining growth. Selection pressure was applied as the pH increased, favoring strains that showed robust growth under alkaline conditions.

This method leveraged natural selection to optimize phenotypes, enhancing both growth and PHB production in alkaline environments. Regular monitoring of growth rates and cell morphology confirmed that Synechocystis adapted to these conditions with minimal impact on cellular structure, retaining its physiological integrity even at pH 11.0. However, growth was significantly inhibited at pH levels of 11.5, which defined an upper threshold for successful adaptation.

## 2. PHB Production Screening and Optimization

PHB production was quantified across a range of pH levels using a fluorescence-based assay. Synechocystis cells were stained with Nile red, a fluorescent dye that binds to PHB granules, allowing for the visualization and quantification of PHB accumulation. Fluorescent intensities correlated with PHB content, establishing an efficient screening process to identify high-PHB-producing strains.

Results indicated that PHB production was highest at pH 11.0, reaching 31.3 wt%, significantly higher than the 20.0 wt% observed at pH 7.5. This improvement was attributed to enhanced metabolic activity and increased availability of bicarbonate ions under moderately alkaline conditions. At pH 11.5, however, PHB content dropped to 17.3 wt%, suggesting a stress-induced reduction in biosynthesis at extreme pH levels.

## 3. RNA-seq Analysis for Transcriptional Insights

To further understand the genetic basis for enhanced PHB production, RNA sequencing was conducted on Synechocystis strains grown at pH 7.5 and pH 11.0. Differential gene expression analysis revealed significant changes in metabolic pathways linked to PHB synthesis. Specifically, genes involved in sugar metabolism (e.g., mannose-1-phosphate guanyltransferase), energy storage (e.g., polyphosphate kinase), and the pentose phosphate pathway (e.g., transketolase) were upregulated in high pH conditions.

Conversely, genes associated with photosynthesis and protein synthesis were downregulated, indicating a shift in cellular resource allocation toward energy storage (PHB production) as a stress response to alkaline conditions. These findings suggest that high pH induces metabolic reprogramming in Synechocystis, favoring bioplastic production as a survival mechanism under stress.

# Building Process

The development of our engineered Synechocystis sp. PCC 6803 for enhanced polyhydroxybutyrate (PHB) production under high pH conditions was a multi-phase process that integrated experimental evolution, synthetic biology techniques, and molecular biology methods.

## 1. Strain Preparation and Cultivation

We began by sourcing a wild-type strain of Synechocystis sp. PCC 6803, a unicellular freshwater cyanobacterium known for its natural ability, to produce PHB and capture atmospheric CO2 via photosynthesis. The initial cultures were grown in a modified BG-11 medium at pH 7.5 under standard laboratory conditions (light intensity, temperature, and CO2 concentration). The goal was to establish a baseline growth curve and PHB production levels under neutral pH conditions before subjecting the cells to evolutionary pressure for high pH adaptation.

![](https://static.igem.wiki/teams/5404/eng-fg1.png)

[Figure 1. A modified BG11 medium without phosphorus and nitrogen was used to enhance the PHB production.]

## 2. Adaptive Laboratory Evolution (ALE) Setup

To drive the evolutionary adaptation of Synechocystis to high pH environments, we designed a gradual pH-incremental system. The process started by cultivating cells at pH 7.5 and then gradually increasing the pH of the growth medium by 0.1 units per cycle. After each pH increase, the cultures were allowed to grow and adapt until stable growth was observed at the new pH level. This approach mimicked natural selection in a controlled laboratory environment, favoring phenotypes that could survive and thrive in progressively alkaline conditions.

We regularly monitored cell growth using optical density (OD750), and cell morphology was assessed using brightfield microscopy. Growth curves were plotted to determine the optimal pH range for both growth and PHB production. Over time, this process resulted in Synechocystis strains that could tolerate pH levels up to 11.5, although optimal PHB production was observed at pH 11.0.

![](https://static.igem.wiki/teams/5404/eng-fg2.png)

[Figure 2. Synechocystis sp. growing at pH 10.]

## 3. PHB Production Assay

After the ALE process, we shifted our focus to identifying the evolved strains that exhibited enhanced PHB production. We used a fluorescence-based quantification assay to screen for PHB-producing strains. Synechocystis cells were stained with Nile red dye, which selectively binds to PHB granules, enabling visualization under a fluorescence microscope. This assay allowed us to quantify intracellular PHB content by measuring fluorescence intensity, which was then correlated with actual PHB production via chloroform-methanol extraction and gravimetric analysis.

Using this screening method, we identified the most promising strains capable of producing high levels of PHB at pH 9.5 to 11.0. The results demonstrated a significant increase in PHB content under moderately alkaline conditions, validating the success of the ALE approach.

![](https://static.igem.wiki/teams/5404/eng-fg3.png)

[Figure 3. PHB extracted (white particles) from the Synechocystis sp. pH7.5 (left) and pH11 (right)]

## 4. Molecular Engineering and Genetic Analysis

To further understand the genetic and molecular mechanisms underlying the enhanced PHB production, we performed RNA sequencing (RNA-seq) on Synechocystis strains grown at both pH 7.5 and pH 11.0. This step involved:

* **RNA Extraction**: We used Trizol reagent to extract total RNA from the cultured cells.
* **Library Preparation**: The RNA samples were prepared using the Illumina TruSeq RNA library kit, and the quality and quantity of the libraries were verified before sequencing.
* **Sequencing and Data Analysis**: The libraries were sequenced using the Illumina platform, and the raw data was processed for quality control and alignment to the Synechocystis reference genome. Differentially expressed genes (DEGs) were identified, and pathway analysis was conducted to link gene expression changes to PHB biosynthesis.

The RNA-seq analysis revealed significant upregulation of genes involved in carbon metabolism, energy storage, and biosynthetic pathways, providing insights into the metabolic reprogramming of Synechocystis in high pH conditions. Also, the downregulation of genes involved in photosynthesis indicates the high pH induced the cellular stress in Synechocystis sp.

![](https://static.igem.wiki/teams/5404/eng-fg4.png)

[Figure 4. Gene-Enrichment and Functional Annotation Analysis using KEGG pathway identifies four significant pathways in both downregulated and upregulated genes.]

# Test

To validate the effectiveness of our engineered Synechocystis sp. PCC 6803 for enhanced polyhydroxybutyrate (PHB) production under high pH conditions, a series of tests were performed across three key areas: growth tolerance, PHB production, and genetic response.

1. **Growth Tolerance Under High pH Conditions**
   * **Objective**: Assess the evolved Synechocystis sp. strains' ability to grow in increasingly alkaline environments.
   * **Method**: Cultures were grown in modified BG-11 medium, and the pH was gradually increased from 7.5 to 11.5 in 0.1-unit increments, following the Adaptive Laboratory Evolution (ALE) method.
   * **Measurements**: Growth was monitored by measuring optical density at 750 nm (OD750), with stabilization times recorded at each pH increment.
   * **Results**: Synechocystis strains showed robust growth up to pH 11.0, with a significant reduction in growth observed at pH 11.5. Optimal growth and stability were achieved at pH 11.0, confirming the successful adaptation of the strains to high pH environments.
2. **PHB Production Screening**
   * **Objective**: Quantify PHB production in Synechocystis strains across different pH levels.
   * **Method**: Nile red dye was used to stain the cells, allowing for fluorescence-based quantification of PHB granules. Fluorescence intensity was correlated with PHB production, confirmed through chloroform-methanol extraction and gravimetric analysis.
   * **Results**: Strains grown at pH 11.0 produced the highest PHB content, reaching 31.3 wt%, a significant improvement over the 20.0 wt% observed at pH 7.5. PHB content decreased to 17.3 wt% at pH 11.5, indicating that extreme pH levels induced stress, reducing biosynthetic capacity.
3. **RNA-seq Transcriptional Analysis**
   * **Objective**: Identify genetic changes contributing to enhanced PHB production under alkaline conditions.
   * **Method**: RNA sequencing was performed on Synechocystis strains grown at pH 7.5 and pH 11.0. Total RNA was extracted, sequenced using the Illumina platform, and analyzed for differential gene expression.
   * **Results**: RNA-seq analysis revealed upregulation of genes related to carbon metabolism, energy storage, and PHB biosynthesis under high pH conditions. In contrast, downregulation of genes involved in photosynthesis suggested a shift in resource allocation towards PHB production, likely as a survival response to stress.

These tests confirm that our engineered Synechocystis sp. PCC 6803 not only thrives in high pH environments but also exhibits significantly enhanced PHB production. The combination of phenotypic adaptation and genetic reprogramming underscores the effectiveness of the engineering approach.

# Learn

Through the 'Build' and 'Learn' phases of our Engineering Cycle, we actively engaged with the core principles of iGEM and Synthetic Biology, iterating and optimizing our approach to enhance PHB production in Synechocystis sp. under high pH conditions.

**Learn #1: Address project challenges using synthetic biology tools and experimental techniques to meet expected outcomes.**

Our goal was to enhance polyhydroxybutyrate (PHB) production in Synechocystis sp. PCC 6803 while ensuring its ability to thrive under high pH conditions. We addressed key project challenges and achieved our objectives through the following:

* **Build**: Implemented Adaptive Laboratory Evolution (ALE) by gradually increasing the culture pH from 7.5 to 11.5 to select for strains with enhanced alkaline tolerance.
* **Build**: Regularly monitored cell growth (via OD750) and assessed cell morphology using brightfield microscopy to ensure physiological integrity during the adaptation process.
* **Build**: Used Nile red staining and fluorescence microscopy to screen for high PHB-producing strains under varying pH conditions.
* **Test**: Quantified PHB production through fluorescence-based assay, confirmed with chloroform-methanol extraction and gravimetric analysis.
* **Test**: Performed RNA sequencing (RNA-seq) to identify differentially expressed genes involved in carbon metabolism and PHB synthesis at pH 7.5 and 11.0.

Our experiments validated that the adapted Synechocystis strains exhibited enhanced PHB production at pH 11.0, with the highest PHB content recorded at 31.3 wt%, compared to 20.0 wt% at pH 7.5. Additionally, RNA-seq analysis revealed the upregulation of genes linked to energy storage and PHB biosynthesis, confirming the success of our engineering approach in optimizing PHB production under alkaline stress.

**Learn #2: Identify and document improvements for the next iteration of the engineering cycle.**

While our first iteration yielded promising results, several limitations emerged:

* **PHB production decline at extreme pH**: We observed a decrease in PHB content at pH 11.5, likely due to stress-induced metabolic disruption. In the next iteration, we would explore co-culturing Synechocystis with symbiotic bacteria that could mitigate stress and support PHB biosynthesis at extreme pH levels.
* **Gene expression variability**: Although RNA-seq provided insights into differentially expressed genes, we could not quantify the precise contribution of each gene to PHB production. A future approach could involve CRISPR-based gene editing to individually modulate key genes, helping us better understand their role in the metabolic reprogramming of Synechocystis.

These refinements will improve our understanding of how Synechocystis responds to high pH stress and could lead to further optimization of PHB production for industrial applications.

**Learn #3: Design and build a new Part, measure its performance, document whether it worked or not, and propose how the results would inform the next design or steps.**

In this project, we contributed two new Parts to the iGEM Registry of Standard Biological Parts, specifically focusing on genes that are differentially expressed in Synechocystis sp. PCC 6803 under high pH conditions. These Parts consist of both upregulated and downregulated differentially expressed genes (DEGs) responsible for enhanced PHB production under alkaline stress.

* **Upregulated DEGs in Synechocystis sp. Under pH11 Conditions** ([Part:BBa\_K5404000 - parts.igem.org](https://parts.igem.org/Part%3ABBa_K5404000))
* **Downregulated DEGs in Synechocystis sp. Under pH 11 Conditions** ([Part:BBa\_K5404001 - parts.igem.org](https://parts.igem.org/Part%3ABBa_K5404001))

These DEGs were identified through RNA-seq analysis and include genes involved in carbon metabolism, PHB biosynthesis, and energy storage pathways, such as mannose-1-phosphate guanyltransferase and polyphosphate kinase. By documenting these genes as Parts, we provide the iGEM community with potential targets for engineering high PHB production in Synechocystis or other organisms.

### **Future Implications:**

* **Application in Synthetic Biology**: The upregulated genes could be used as potential overexpression targets for other cyanobacteria or algae species, broadening the scope of organisms capable of high PHB production. Conversely, the downregulated genes provide insight into metabolic pathways that may need to be repressed to optimize PHB biosynthesis under stress conditions.
* **Further Testing and Iteration**: Future iterations will involve validating the specific contribution of each gene to PHB production through CRISPR-based knockouts or overexpression in Synechocystis. This will allow us to fine-tune the genetic network for even higher yields of PHB.
* **Industrial Applications**: These Parts pave the way for the development of biotechnological solutions to scale PHB production for industrial applications. The ability to engineer robust, high-PHB-producing strains that can thrive under various environmental conditions, particularly in alkaline environments, could be a game-changer for sustainable bioplastics.

In summary, these newly designed Parts contribute valuable genetic tools for future synthetic biology projects, opening pathways for optimizing PHB production and expanding the use of Synechocystis as a bioplastic-producing organism in industrial contexts.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

Experiments

# Experiments Meetings

## Experiments Day 1

### **Agenda 1** : Practicing with Micropipettes

**Experimental Principle**

* Accuracy in micropipette practice is crucial for making the medium correctly without any mistakes.

**Materials Needed**

1. Micropipette
2. Pipette tips
3. Micro test tubes
4. 600 mL Beaker filled with distilled water
5. Disposal Bin

**Procedure**

1. Add the correct pipette tip onto the micropipette. Ensure that it is put on tightly and does not fall.
2. Fully press the button on the top of the micropipette and place it into the beaker. Make sure that only the tip comes in contact with the water.
3. Release the button to absorb the liquid. Confirm that the correct amount of water has been absorbed.
4. Take the micropipette out of the water and place it over the micro test tube.
5. Press the button on the top of the micropipette once more and make sure all the water is released.
6. Release the tip of the micropipette into a disposal bin by pressing the button on the side.

![](https://static.igem.wiki/teams/5404/exp-fg1.png)

[Figure 1: Korea-HS Member Practicing Micropipette Ssage by Emptying Out Distilled Water into a Micro Test Tube]

### **Agenda 2** : Preparation of Stock Solutions

**Experimental Principle**

* Concentrated stock solutions are prepared to create the final modified BG11 medium.
* Nitrogen and phosphorus sources will be eliminated from the sources to ensure PHB production in bacteria.

**Materials Needed**

1. NaNO3 (Sodium Nitrate)
2. K2HPO4 (Dipotassium Phosphate)
3. MgSO4·7H2O (Magnesium Sulfate Heptahydrate)
4. CaCl2·2H2O (Calcium Chloride Dihydrate)
5. Citric Acid·H2O
6. Ferric Ammonium Citrate
7. Na2EDTA·2H2O (Disodium EDTA)
8. Na2CO3 (Sodium Carbonate)
9. Electronic balance
10. Spatulas
11. Weighing boats
12. 50 mL screw-capped tubes
13. Distilled water
14. 100 mL graduated cylinder
15. Disposal Bin
16. Test tube rack

**Procedure**

* For each chemical:

1. Add the weighing boat into an electronic balance and zero it.

   ![](https://static.igem.wiki/teams/5404/exp-fg2.png)

   [Figure 2: Korea-HS Member Getting the Correct Amount of Mass of the Powder Using the Electronic Balance]
2. Add the correct amount of powder onto the weighing boat using a spatula. Make sure the weight is as close to the desired value as possible. If there is any excess material, place it in the disposal bin.
3. Empty all of the powder on the weighing boat into a 50 mL tube.

   ![](https://static.igem.wiki/teams/5404/exp-fg3.png)

   [Figure 3: Korea-HS Member Emptying Out the Powder Using a Spatula]
4. Label the chemical using a Sharpie. Include the date, name of the chemical, and final concentration.
5. Get 50 mL of distilled water from the water distiller using a 100 mL graduated cylinder.

   ![](https://static.igem.wiki/teams/5404/exp-fg4.png)

   [Figure 4: Korea-HS Member Getting Distilled Water from the Water Distiller]
6. Pour all of the distilled water in the graduated cylinder into the 50 mL tubes.

   ![](https://static.igem.wiki/teams/5404/exp-fg5.png)

   [Figure 5: Korea-HS Member Adding 50 mL of Distilled Water into the Test Tube]
7. Close the lid of the 50 mL tube, then constantly flip it for one minute to ensure that it is thoroughly mixed.
8. Place the tube into a test tube rack. Make sure the labeled side is faced forward.

   ![](https://static.igem.wiki/teams/5404/exp-fg6.png)

   [Figure 6: All of the Stock Solutions Labeled and Placed onto the Test Tube Rack]

### Agenda 3: Creating of Modified BG11 Medium

**Experimental Principle**

* Stock solutions are combined in proportions to create the final culture medium.
* The medium will be prepared  **without** nitrogen and phosphorus to promote PHB production.

**Materials Needed**

1. Prepared stock solutions
2. Pipette aid
3. Pipette aid tips
4. 500 mL bottle
5. Graduated Cylinder
6. Second test tube rack

**Procedure**

1. For each stock solution:
   1. From the test tube rack, get one of the stock solutions.
   2. Get a pipette aid, and place a new pipette aid tip onto the edge.
   3. Using the pipette aid, add 5 mL of the stock solution into the 500 mL bottle.

      ![](https://static.igem.wiki/teams/5404/exp-fg7.png)

      [Figure 7: Korea-HS Member Getting 5 mL of the Stock Solution using a Pipette Aid]
   4. Replace the pipette tip to avoid contamination.
   5. Place the used test tube onto a separate rack to avoid confusion.
2. Once all the stock solutions are correctly added, fill the rest of the container with distilled water.
3. Label the bottle as “modified BG11 medium.”

![](https://static.igem.wiki/teams/5404/exp-fg8.png)

[Figure 8: Completed Bottle of the Modified BG11 Medium]

### **Agenda 4** : Measurement of pH

**Experimental Principle**

* pH is critical for bacterial growth, and therefore we must maintain the pH of the medium at the desired level.

**Materials Needed**

1. pH meter with probe
2. Calibration solutions
3. NaOH solution
4. Distilled water (for rinsing)

**Procedure**

1. Remove the pH probe from the electrode storage bottle.
2. Rinse the probe gently with distilled water and use a paper towel to wipe the probe dry.
3. Place the probe into a 4.0 pH calibration solution, press the calibration button on the pH meter, and wait.
4. Once the meter reads 4.0, remove the probe from the solution and repeat step 2.
5. Place the probe into a 7.0 pH calibration solution, press the calibration button on the pH meter, and wait.
6. Once the meter reads 7.0, remove the probe from the solution and repeat step 2.
7. Place the probe into a 10.0 pH calibration solution, press the calibration button on the pH meter, and wait.
8. Once the meter reads 10.0, remove the probe from the solution and repeat step 2.
9. Prepare the liquid-based medium and place the probe inside the medium.

   ![](https://static.igem.wiki/teams/5404/exp-fg9.png)

   [Figure 9: Image of the pH Probe Placed Inside of the Liquid Medium]
10. Once the pH stabilizes, take note of the pH, and then repeat step 2

    ![](https://static.igem.wiki/teams/5404/exp-fg10.png)

    [Figure 10: Image of the Final pH of our Liquid Medium Shown on the pH Meter]
11. Place the pH probe back into the electrode storage bottle.

## Experiments Day 2

### **Agenda 5**: CO2 Capture and Conversion using Synechocystis and RuBisCo

**Experimental Principle**

* **Direct Capture**  and  **Conversion of CO2**
   from the air into 3-PGA using bacteria and the enzyme RuBisCo in the Calvin Cycle.
  + Cultivate the bacteria at a high pH
  + Prepare a
     **solid culture plate**
  + Apply the cultured synechocystis
  + Use the
     **streaking method**
     to spread the bacteria to the agar plate with a modified BG11 medium

**Materials Needed**

1. 1 electronic weighing balance
2. 1 spatula
3. 2 weighing boats
4. 1 200mL measuring cylinder
5. 1 microwave
6. 10 sterile petri plates (1 per person)
7. 1 alcohol lamp
8. 1 timer
9. 2.8g of nutrient agar powder
10. 1 200mL of BG11 solution
11. 1 200μL pipette
12. 1 glass rod
13. 1 ethanol spray
14. 1 glass bottle
15. 10 tissue papers
16. 1 400mL of distilled water
17. 1 centrifuge
18. 1 shaking incubator
19. 1 500mL conical flask

**Procedure**

1. **Agar-BG11 mixture Preparation**
   1. Measure 2.8g of agar powder using an electronic weighing balance.
      1. If there are excesses in case of spillage/loss when pouring, discard the excess agar powder after measuring mass into an empty weighing boat.
   2. Pour the 2.8g of agar powder into a separate glass bottle using a spatula to mix.
   3. Measure 200mL of Bg11 medium using a measuring cylinder.
   4. Pour 200mL of the Bg11 medium into the glass bottle that contains the agar powder.
   5. Mix the solution until it seems hazy and shows a yellowish mixture.
   6. Seal the top of the glass bottle with foil (make sure the top is completely covered).
   7. Microwave the solution for 2-3 minutes, stop immediately if the solution boils or overflows.
      1. Repeat this step by stirring the solution in the middle until the solution becomes clear.

   ![](https://static.igem.wiki/teams/5404/exp-fg11.png)

   [Figure 11: Korea-HS Team Member Pouring 200mL of Modified Bg11 into the Glass Bottle with 2.8g of Agar Powder]
2. **Agar Plates Preparation**
   1. Label each sterile petri plate with the name, date, colony number, and the start date of culture on the bottom edge.
   2. Wrap the glass bottle with 2-3 tissue papers as it is very hot and to avoid burns.
   3. Pour approximately 15mL (till when the solution fills the whole plate) of molten agar-Bg11 mixture into each sterile Petri plate.
      1. Pour it very carefully so that you avoid creating bubbles.
   4. Partially close the lid (half) and leave it for 15 minutes to solidify under a sterile environment with the alcohol lamp.

      ![](https://static.igem.wiki/teams/5404/exp-fg12.png)

      [Figure 12: Korea-HS TeamMmember Pouring 15mL of Molten Agar-Bg11 Mixture into the Sterile Petri Plate]
3. **Colony Centrifuge**
   1. First, place the bacterial culture tubes into a centrifuge at 3500 rpm for 5 minutes to concentrate the bacteria.
   2. Use a pipette set to 200μL to pump the bacteria culture and distribute them evenly by pipetting up and down 5-10 times.

      ![](https://static.igem.wiki/teams/5404/exp-fg13.png)

      [Figure 13: Korea-HS Team Member Pumping Bacteria Culture with a Pipette to Distribute Them Evenly]
   3. After that, pull out the solution and drop it on the surface of the plate (draw like a circle).
      1. Make sure to remove the pipette tip after use.
   4. Sanitize the glass rod by spraying ethanol 2-3 times and wiping it with tissue paper.
      1. Repeat this every time you use it once for each bacterial culture.
   5. Use a glass rod for streaking, and rub it against the surface of the plate.
      1. Rotate the plate while streaking to ensure even distribution of the bacteria.

      ![](https://static.igem.wiki/teams/5404/exp-fg14.png)

      [Figure 14: Korea-HS Team Member Rubbing the Glass Rod Against the Surface of the Plate for Streaking]
   6. When complete, close the lid and flip the plate.
      1. Incubate at 30ºC for 1 week - 3-4 plates stacked at once, place tube inside shaking incubator.

      ![](https://static.igem.wiki/teams/5404/exp-fg15.png)

      [Figure 15: All Sterile Petri Plates Placed in an Incubator Maintained at 30ºC]

## Experiments Day 3

### **Agenda 6** : Development and Optimization of Synechocystis species Strain

**Experimental Principle**

* Familiarizing with Microbiological Techniques
  + Micropipetting
  + Colony picking
  + Cell imaging* The primary objective of this experiment is to gain proficiency in the essential microbiological techniques listed above. The attained skills will facilitate the development and optimization of a strain of Synechocystis that can efficiently capture and convert CO2 from the atmosphere.

**Materials Needed**

1. Cell Imaging Microscope
2. Bacteria Culture Kit
3. Micropipette
4. Modified BG11 Medium

**Procedure**

1. Insert a tip at the front of the micropipette.
2. Put the solution in the tube for the bacteria sample.
3. Poke the bacteria 3-4 times and soak it in and out inside the tube using the micropipette to ensure the bacteria is in the tube.

   ![](https://static.igem.wiki/teams/5404/exp-fg16.png)

   [Figure 16: Kor-HS Team Member Poking the Bacteria with a Micropipette]
4. Inoculate Synechocystis cells into the modified BG11 medium, designed to sustain high pH levels.
5. Periodically sample and streak cells onto solid BG11 medium to isolate single colonies.
6. Select colonies exhibiting altered coloration or other phenotypic changes indicative of potential adaptive mutations.

   ![](https://static.igem.wiki/teams/5404/exp-fg17.png)

   [Figure 17: Kor-HS Team Member Creating Colonies with a Micropipette]
7. Select the strains that show increased PHB production, identifiable by increased granule formation within cells or using chemical assays that quantify PHB content.
8. Image selected Synechocystis species that can grow in high pH and produce PHB efficiently
    .

   ![](https://static.igem.wiki/teams/5404/exp-fg18.png)

   [Figure 18: Kor-HS Team Member Looking into the Microscope to See Synechocystis sp.]

## Experiments Day 4

### **Agenda 6** : Stain Synechocystis cells with Nile Red for visualization of PHB

**Materials Needed**

1. Synechocystis sp. culture
2. Nile Red stain (Sigma-Aldrich, N3013)
3. Dimethyl sulfoxide (DMSO) or ethanol (for Nile Red stock solution)
4. Phosphate-buffered saline (PBS)
5. Glass slides
6. Coverslips
7. Fluorescent microscope
8. Pipettes and tips
9. Microcentrifuge tubes
10. Vortex mixer

**Procedure**

1. **Preparation of Nile Red Stock Solution**
   1. Dissolve Nile Red in DMSO or ethanol to prepare a 1 mg/mL stock solution.
   2. Store the stock solution at room temperature in a dark container to protect it from light.
2. **Culture Preparation**
   1. Grow Synechocystis cells in a modified BG11 growth medium until they reach the desired density (typically in the exponential growth phase).
   2. Harvest the cells by centrifugation at 5000 x g for 5 minutes.
   3. Discard the supernatant and resuspend the cell pellet in PBS to wash the cells.
   4. Centrifuge again at 5000 x g for 5 minutes and discard the supernatant.
   5. Resuspend the washed cell pellet in PBS to the desired concentration (OD750 of 0.5).

   ![](https://static.igem.wiki/teams/5404/exp-fg19.png)

   [Figure 19: Kor-HS Team Member Adding a Plate into the Centrifuge]
3. **Staining Procedure**
   1. Take 1 mL of the resuspended Synechocystis cells in a microcentrifuge tube.
   2. Add 5 µL of Nile Red stock solution to the cell suspension (final concentration of 5 µg/mL).
   3. Vortex briefly to mix.
   4. Incubate the mixture in the dark at room temperature for 10-15 minutes.

   ![](https://static.igem.wiki/teams/5404/exp-fg20.png)

   [Figure 20: Kor-HS Team Member Inserting Nile Red]
4. **Washing**
   1. After incubation, centrifuge the stained cells at 5000 x g for 5 minutes.
   2. Discard the supernatant and wash the cell pellet with 1 mL of PBS to remove excess stain.
   3. Centrifuge again at 5000 x g for 5 minutes and discard the supernatant.
   4. Resuspend the cell pellet in 100 µL of PBS for microscopy.
5. **Microscopy**
   1. Place a small drop (10 µL) of the stained cell suspension onto a clean glass slide.
   2. Gently place a cover slip over the drop.
   3. Observe the stained cells under a fluorescent microscope using appropriate filter settings (excitation at 488 nm and emission at 600 nm).
   4. Capture images.

   ![](https://static.igem.wiki/teams/5404/exp-fg21.png)

   [Figure 21: Kor-HS Team Member Preparing for Microscopy]

   ![](https://static.igem.wiki/teams/5404/exp-fg22.png)

   [Figure 22: Kor-HS Team Member Looking into the Stained PHB]

### Protocol for Quantifying Synechocystis Cell Number Using a Microplate Reader

**Objective**
: To quantify the number of Synechocystis cells using a microplate reader by measuring optical density (OD) at 750 nm.

**Materials Needed**

1. Synechocystis sp. culture
2. Phosphate-buffered saline (PBS)
3. 96-well microplate (clear, flat-bottom)
4. Pipettes and tips
5. Microplate reader capable of measuring absorbance at 750 nm
6. Sterile cuvettes (for calibration curve preparation)
7. Spectrophotometer (for calibration curve preparation)

**Procedure**

1. **Preparation of Synechocystis Culture**
   1. Grow Synechocystis cells in BG11 growth medium until they reach the desired density (typically in the exponential growth phase).
2. **Preparation of Serial Dilutions**
   1. Prepare a series of serial dilutions of the Synechocystis culture to create a calibration curve. Typically, prepare dilutions such as 1:2, 1:4, 1:8, 1:16, and so on, using PBS.
   2. Transfer 1 mL of each dilution into sterile cuvettes.
3. **Measurement of Optical Density for Calibration Curve**
   1. Measure the OD at 750 nm of each dilution using a spectrophotometer.
   2. Record the OD values and corresponding cell concentrations (cells/mL) for each dilution.
   3. Plot the OD values against cell concentrations to create a standard calibration curve.
4. **Sample Preparation**
   1. Harvest the Synechocystis cells by centrifugation at 5000 x g for 5 minutes.
   2. Discard the supernatant and resuspend the cell pellet in PBS.
   3. Adjust the cell suspension to an appropriate concentration range based on the calibration curve.
5. **Loading the Microplate**
   1. Transfer 100 µL of each sample and standard (serial dilutions) into the wells of a 96-well microplate. Ensure to include PBS as a blank.
   2. It is recommended to perform each measurement in triplicate for accuracy.
6. **Measurement Using Microplate Reader**
   1. Measure the absorbance of each well at 750 nm using the microplate reader.
   2. Ensure the microplate reader is set to the correct wavelength (750 nm).

   ![](https://static.igem.wiki/teams/5404/exp-fg23.png)

   [Figure 23: Placing a Plate into the Quantification Machine]
7. **Data Analysis**
   1. Subtract the blank (PBS) absorbance value from all sample absorbance values to correct for background absorbance.
   2. Use the calibration curve to convert the corrected OD750 values of the samples to cell concentrations (cells/mL).
8. **Calculation**
   1. If needed, calculate the total number of cells in your original sample by multiplying the concentration (cells/mL) by the total volume of your culture.

## Experiments Day 5

### **Agenda 7** : PHB Extraction from Synechocystis

**Experimental Principle**

* The primary objective was to extract PHB from the Synechocystis bacteria.
  + Utilize Synechocystis cells that reached their stationary phase to maximize PHB accumulation
  + Use sodium nitrite to lyse cells and release PHB granules
  + Chloroform was used to dissolve the PHB, allowing it to be extracted.
  + The final product was stored and purified for further use.

**Materials Needed**

1. Synechocystis culture
2. Chloroform
3. Sodium Nitrate (NaNO2)
4. Ethanol (4 degrees Celcius)
5. Centrifuge
6. Water bath or shaker
7. Glass vials or tubes
8. Micropipettes
9. Micropipette tips
10. Analytical Balance
11. Drying oven or desiccator
12. Protective glass screen

**Procedure**

1. **Cell Preparation**
   1. Grow Synechocystis cells under suitable conditions until they reach the stationary phase for higher PHB accumulation.
   2. Harvest the cells by centrifuging the culture at 5000 x g for 10 minutes.

   ![](https://static.igem.wiki/teams/5404/exp-fg24.png)

   [Figure 24: Korea-HS Team Member Centrifuging the Culture]
2. **Cell Lysis**
   1. Resuspend the cell pellet in a suitable volume of distilled water.
   2. Add 1% (w/v) sodium nitrite (NaNO₂) to the cell suspension.
   3. Incubate the mixture at 36.5°C for 1-2 hours to facilitate cell lysis and release of PHB granules. Must work quickly to transfer samples into tubes to prevent evaporation.
3. **PHB Extraction**
   1. After cell lysis, add chloroform to the suspension in a 1:1 ratio (v/v).

      ![](https://static.igem.wiki/teams/5404/exp-fg25.png)

      [Figure 25: Korea-HS Team Member Adding Chloroform to the Suspension]
   2. Vigorously mix or shake the solution for 30 minutes to allow PHB to dissolve in the chloroform phase. Use a micropipette set to 300 microL to inject chloroform, replacing the tip after each use to avoid damage.
4. **Phase Separation**
   1. Centrifuge the mixture at 5000 x g for 10 minutes to separate the chloroform layer containing PHB from the aqueous phase.
   2. Carefully collect the bottom chloroform phase using a micropipette. Pull down slowly to avoid disturbing the layers.

      ![](https://static.igem.wiki/teams/5404/exp-fg26.png)

      [Figure 26: Korea-HS Team Member Removing the Bottom Layer of the Chloroform Phase using a Micropipette]
   3. Remove the top layer of the solution as well.
5. **Precipitation of PHB**
   1. To precipitate PHB, add an excess of cold ethanol (4°C) to the chloroform extract.
   2. Let the mixture stand at low temperature for several hours

   ![](https://static.igem.wiki/teams/5404/exp-fg27.png)

   [Figure 27: Kor-HS Team Member Precipitating Ethanol]

   ![](https://static.igem.wiki/teams/5404/exp-fg28.png)

   [Figure 28: Images of samples after precipitation]
6. **Collection and Purification**
   1. Centrifuge the mixture again to collect the PHB precipitate.
   2. Wash the precipitate with ethanol or water to remove impurities.
   3. Dry the PHB in a drying oven at 50-60°C or under a vacuum in a desiccator.
7. **Quantification**
   1. Weigh the dried PHB to determine the yield.
8. **Storage**
   1. Store the purified PHB in a cool, dry place until further use.

### **Agenda 8** : Analyzing the Cytotoxicity of PHB on Human Skin Cells

**Experimental Principle**

* The aim is to assess the cytotoxic effects of extracted PHB on human skin cells.
* PHB samples are placed in a non-toxic solvent to ensure safe exposure to cells.
* The PrestoBlue™Cell Viability Assay is used to measure metabolic activity to show cell heath.
* A dose-response curve is created to find the relationship between PHB concentration and cell viability.

**Materials Needed**

1. PHB samples
2. Human skin cells
3. Dulbecco’s Modified Eagle Medium (DMEM)
4. Fetal bovine serum (FBS)
5. Penicillin-streptomycin solution
6. Phosphate-buffered saline (PBS)
7. PrestoBlue™ Cell Viability Reagent
8. Trypsin-EDTA solution
9. Incubator (37°C, 5% CO₂)
10. Centrifuge
11. Microplate reader

**Procedure**

1. **Preparation of PHB Samples**
   1. Dissolve the PHB in chloroform or DMSO and ensure that the solvent concentration is non-toxic.
   2. Prepare the PHB concentrations by serial dilution in the culture medium and sterilize by filtering through a 0.22 µm filter.
2. **Cell Culture**
   1. Cultivate human skin cells in DMEM with 10% FBS and 1% penicillin-streptomycin.
   2. Incubate cells at 37 degrees Celcius in a humidified atmosphere with 5% CO2.
   3. When cells reach 70-80% confluence, detach them using trypsin-EDTA.
   4. Add trypsin using a micropipette to degrade the protein membrane from the cells.
   5. Incubate the cell culture with trypsin for 5 minutes in the CO2 incubator at 37 degrees Celsius.
   6. Centrifuge the detached cells at 300 x g for 5 minutes.
   7. Resuspend the cell pellet in a fresh culture medium.

   ![](https://static.igem.wiki/teams/5404/exp-fg29.png)

   [Figure 29: Centrifuging Process of Detached Cells]
3. **Seeding Cells in 96-Well Plates**
   1. Use a micropipette to see the cells into 96-well plates at a density of 5,000-10,000 cells per well in 100µL of complete medium.
   2. Allow the cells to adhere and grow for 24 hours before treatment.

   ![](https://static.igem.wiki/teams/5404/exp-fg30.png)

   [Figure 30: Kor-HS Team Member Adding Skin Cells into a 96 Well Plate]
4. **Treatment with PHB**
   1. Replace the culture medium with 100 µL of fresh medium containing various PHB concentrations.
   2. Include negative control (cells with culture medium only) and positive control (known cytotoxic agent).
   3. Incubate cells with PHB for 24 to 72 hours.
5. **PrestoBlue™ Assay for Cytotoxicity**
   1. Add 10 µL of PrestoBlue™ reagent to each well.
   2. Gently mix the plate for even distribution.
   3. Incubate at 37°C for 1 to 2 hours (1 hour is typically sufficient).

   ![](https://static.igem.wiki/teams/5404/exp-fg31.png)

   [Figure 31: Kor-HS Member Adding PrestoBlue into a 96 Well Plate]
6. **Measurement and Data Analysis**
   1. Measure fluorescence (excitation 560 nm, emission 590 nm) or absorbance (570 nm).
   2. Normalize readings by subtracting background.
   3. Calculate cell viability percentage relative to control.
   4. Plot the dose-response curve and determine IC50 if applicable.
7. **Statistical Analysis**
   1. Perform ANOVA followed by post hoc tests (e.g., Tukey's test) for multiple comparisons.

## Experiments Day 6

### **Agenda 9** : PHB Cytotoxicity Test Under Modified Lab Conditions

**Experimental Principle**

* To quantify and analyze the extent of PHB cytotoxicity and synthesis under various conditions fostered through previous labs.
  + Evolutionary selection to identify strains with enhanced PHB production (reverse approach) differentiates the results extracted from conventional methods.
  + **PrestoBlue™ Assay**
     was conducted with a spectrophotometer to determine the cytotoxicity.
  + **ANOVA test**
     was performed as a statistical test to assess the significance of the results.

**Materials Needed**

1. Centrifuge
2. Oven
3. Test tubes
4. Electronic balance
5. Incubator
6. Distilled Water
7. PrestoBlue
8. PHB culture medium
9. Micropipettes
10. Micropipettes tips

**Procedure**

1. Wash each test tube 3 times using 500 microliters of distilled water.
2. Vortex each test tube after each wash to remove impurities.
3. Wash PHB with distilled water.

   ![](https://static.igem.wiki/teams/5404/exp-fg32.png)

   [Figure 32: Kor-HS Team Member Adding Water to Wash PHB]
4. Centrifuge the PHB in test tubes at maximum speed in order to collect PHB.
5. Measure the weight of the PHB produced from the cells.

   ![](https://static.igem.wiki/teams/5404/exp-fg33.png)

   [Figure 33: Weighing the Centrifuged Test Tube]

   1. The test tubes were placed in a dry oven to evaporate all water, ensuring accurate weight measurements.
   2. To determine the weight of the PHB, the weight of 4 empty test tubes was first measured and recorded.
   3. Then, PHB was added to each of the test tubes, and the weight of the tubes with PHB was recorded.
   4. The weight of the PHB was obtained by subtracting the weight of the empty tube from the weight of the tube with PHB.
6. Take the image and compare the weight of PHB produced from different conditions.
7. After 24 hours, replace the culture medium with 100 µL of fresh medium containing the various concentrations of PHB.
   1. Include a negative control (cells with culture medium only) and a positive control (cells treated with a known cytotoxic agent).
      1. The negative control provides a baseline measurement for cell viability and growth in the absence of PHB.
      2. The positive control ensures that the assay can detect cytotoxic effects by using a known cytotoxic agent; it validates the sensitivity and reliability of the assay.
8. Incubate the cells with PHB for 24 to 72 hours, depending on the desired exposure time.
9. After the treatment period, add 10 µL of PrestoBlue™ reagent directly to each well containing 100 µL of cell culture medium.

   ![](https://static.igem.wiki/teams/5404/exp-fg34.png)

   [Figure 34: Kor-HS Team Member Inserting PrestoBlue into a 96 Well Plate]

   1. When adding the reagent, a new micropipette tip was used for each addition to avoid cross-contamination.
   2. The reagent was pipetted gently to the side rather than to the center of the well as it was difficult to sense the depth of the well and therefore minimize the impact of punctures.
   3. The solution (PrestoBlue) is a membrane-permeable chemical. Therefore, when it penetrates the skin cells it creates a reducing condition that turns the blue color into pink.
   4. If the blue color remains, this signifies that there are not a lot of cells (measures for the cell viability) as cells have intact membranes so permeation of the membranes will cause a color change.
   5. Negative control wells (cells with culture medium only) exhibited no color change.

   ![](https://static.igem.wiki/teams/5404/exp-fg35.png)

   [Figure 35: A Full Image of the 96 Well Plate after having PrestoBlue Assay]
10. Gently mix the plate to ensure even distribution of the reagent.
11. Incubate the plate at 37°C for 30 minutes.
    1. During incubation, the metabolically active cells reduce the PrestoBlue™ reagent, causing a color change.
12. Measure the absorbance between 570 nm and 600 nm (using a spectrophotometer, the wavelengths used were 570 nm and 600 nm)

    ![](https://static.igem.wiki/teams/5404/exp-fg36.png)

    [Figure 36: 570nm Absorbance Test Results]

    ![](https://static.igem.wiki/teams/5404/exp-fg37.png)

    [Figure 37: 600nm Absorbance Test Results]
13. Normalize the readings by subtracting the background (e.g., a well with culture medium and PrestoBlue™ but no cells).
    1. Background absorbance (culture medium + reagent without cells) was subtracted from sample readings.
14. Calculate the percentage of cell viability relative to the control (untreated cells) using the following formula:
    1. Cell viability (%) = [(absorbance of sample - background) / [absorbance of untreated control - background)] \* 100
15. Plot the cell viability against the concentration of PHB to generate a dose-response curve. If applicable, determine the IC50 value (the concentration of PHB that reduces cell viability by 50%).
16. Perform statistical analysis to assess the significance of the results, typically using ANOVA followed by post hoc tests (e.g., Tukey's test) for multiple comparisons.

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Results Description

# Research Goal & Overview

Our project aimed to engineer **Synechocystis sp. PCC 6803**, a unicellular freshwater cyanobacteria that produces polyhydroxybutyrate (PHB) by consuming CO₂ from the atmosphere. Specifically, we enhanced the production of PHB and reduction of CO₂ by exposing bacteria under high pH conditions, addressing climate change and plastic pollution. We conducted a series of experiments involving adaptive laboratory evolution (ALE), PHB production screening, and RNA sequencing (RNA-seq) analysis to achieve this goal.

# 1. Adaptive Laboratory Evolution (ALE) for High pH Tolerance

Adaptive Laboratory Evolution (ALE) is a method for naturally selecting suitable phenotypes to cultivate desired characteristics in the laboratory. This method is highly based on Darwin’s theory of natural selection, stating that evolution, or change in genotype and phenotype over generations, can occur to increase the chance of survival. We simulated natural selection by increasing the pH of the growth medium in laboratory conditions, letting the cyanobacteria adapt to high pH conditions and produce a high amount of PHB.

## 1.1. Incremental pH Increase and Selection

We initiated our experiment by cultivating Synechocystis sp. under optimal conditions at pH 7.5, a pH level mainly of H₂O. To promote adaptation to higher pH levels, we employed a pH increment strategy, gradually increasing the pH of the growth medium by 0.1 pH units per cycle. After each incremental adjustment, the cultures were maintained at the new pH level until stable growth was observed. This stepwise approach allowed the cells to adapt gradually to the increasing alkalinity, applying selective pressure to favor strains capable of thriving under those conditions. This gradual pH elevation minimized shock to the cells and facilitated the selection of a phenotype that can produce PHB efficiently, effectively harnessing ALE principles to enhance pH tolerance.

## 1.2. Monitoring Growth and Adaptation

![](https://static.igem.wiki/teams/5404/res-fg1.png)

[Figure 1: Growth curves of Synechocystis sp. during ALE at increasing pH levels. ANOVA (Analysis of variance) was used together with Tukey’s post hoc test to calculate the p-value. Compared to the pH 7.5 sample, pH 10.5, 11, and 11.5 significantly decreased the Synechocystis sp. growth.]

These results illustrate that while Synechocystis sp. is highly adaptable and capable of sustaining growth in a wide pH range, there are limits to this adaptability. The optimal growth occurs between pH 7.5 and 9.5, with substantial growth still observed up to pH 11.0. However, at pH levels beyond 11.0, the extreme alkalinity adversely affects cellular functions, leading to significant growth inhibition. Understanding these pH effects is crucial for optimizing cultivation conditions for applications such as sustainable bioplastic production, where maintaining cell viability and productivity is essential.

![](https://static.igem.wiki/teams/5404/res-fg2.png)

[Figure 2: Increasing the pH does not affect the Synechocystis sp. cell morphology (Cyanobacteria grown at pH 11.5 was not more robust than to bacteria grown at pH 9.5). Brightfield images of cells grown at pH 7.5, pH 9.5, pH 11, and pH 11.5 were presented. Scale bar = 100 µm]

Our study demonstrates that Synechocystis sp. exhibits robust growth and maintains consistent cell morphology in alkaline conditions up to pH 11.0, with only a slight reduction in growth rate compared to neutral pH levels. Brightfield microscopy images of cells grown at pH levels 7.5, 9.5, 11.0, and even 11.5 revealed no significant changes in cell shape, size, or distribution. Figure 2 indicates that increasing the pH does not affect the Synechocystis sp. cell morphology, suggesting that the cells are resilient to high pH stress and can maintain their structural integrity even under more alkaline conditions.

The preservation of cell morphology is crucial as it reflects the cells' ability to sustain essential physiological processes, including photosynthesis and metabolic activities related to polyhydroxybutyrate (PHB) production. Maintaining consistent morphology ensures that the cells remain functional and efficient in converting atmospheric CO₂ into valuable bioplastics.

# 2. Screening for Enhanced PHB Production

![](https://static.igem.wiki/teams/5404/res-fg3.png)

[Figure 3: Fluorescence-based quantification assay for PHB content in Synechocystis cells. (A) Intracellular PHB contents are linearly correlated with fluorescent intensities of Nile-red stained PHB. (B) Fluorescent images of Nile red-stained PHB-producing cells. Scale bar = 50 µm]

The values of fluorescent intensity and intracellular PHB content were plotted on a scatter graph, and a linear regression line was drawn to determine the strength of the correlation. The R² value of 0.9091 of the linear trendline indicates a strong, positive correlation between intracellular PHB content and the fluorescence intensity of Nile red-stained PHB granules (Figure 3A). This confirms that fluorescence intensity serves as an accurate proxy for quantifying PHB levels in Synechocystis cells.

Fluorescence microscopy images captured under varying pH conditions from pH 7.5 to 11.5 further illustrate the distribution and intensity of Nile red-stained PHB granules in PHB-producing Synechocystis cells (Figure 3B). PHB content, normalized to cell dry weight, was quantified using the chloroform-methanol extraction method. As shown in Figure 3B, pH conditions that resulted in higher PHB content – pH 9.5 and 11.0 – displayed fluorescence of visibly greater intensity than those with lower PHB content. Such correlation indicates the reliability and efficiency of Nile red staining in visualizing and quantifying PHB accumulation under different environmental conditions in Synechocystis sp.

![](https://static.igem.wiki/teams/5404/res-fg4.png)

[Figure 4: Increasing pH up to 11.0 increased the PHB content (wt%) in Synechocystis cells.]

Figure 4 demonstrates the effect of varying pH levels on PHB content (wt%) in Synechocystis cells. The PHB content increased as the pH level rose from 7.5 to 11.0, with a significant peak observed at pH 11.0, where the PHB content reached 31.3 wt% (p = 0.002). At pH 7.5 and 9.5, the PHB content was measured at 20.0 wt% and 24.7 wt%, respectively. However, a further increase in pH to 11.5 resulted in a notable decrease in PHB content to 17.3 wt%. These results indicate that moderately alkaline conditions (pH 11.0) optimally enhance PHB production in Synechocystis, whereas more extreme pH conditions (either lower or higher) reduce the accumulation of PHB.

Based on the results from Figure 4, we observed that increasing the pH levels significantly affected PHB production in Synechocystis cells, with a notable peak at pH 11.0, where PHB content reached 31.3 wt%. This suggests that moderately alkaline conditions enhance PHB biosynthesis, likely due to the cells' improved metabolic activity and increased availability of bicarbonate ions for carbon fixation under these conditions. However, at pH levels beyond 11.0, such as 11.5, the extreme alkalinity resulted in a marked reduction in PHB content, dropping to 17.3 wt%. This indicates a threshold beyond which further increases in pH become detrimental to PHB accumulation, likely due to cellular stress that disrupts normal metabolic functions. Overall, these findings support the hypothesis that Synechocystis sp. can be engineered for enhanced PHB production under high pH conditions, but they also highlight the importance of maintaining pH within an optimal range to maximize bioplastic yield.

![](https://static.igem.wiki/teams/5404/res-fg5.png)

[Figure 5: PHB showed no cytotoxicity up to 10% of concentrations on Detroit 551 human fibroblast skin cell line. Cell viability was analyzed using the Prestoblue assay. (Left) The standard curve shows a high correlation between absorbance and number of cells (R2 = 0.991). (Right) The absorbance was measured with different concentrations of PHB (0 to 10%) up to 96 hours. One-way ANOVA test with tukey’s post hoc test was used to calculate p-value.]

The cytotoxicity of Polyhydroxybutyrate (PHB) on Detroit 551 human fibroblast skin cells was evaluated using the Prestoblue assay. The standard curve generated (left graph) displayed a high linear correlation (R² = 0.991) between absorbance values at 570/600 nm and the number of cells, indicating a robust relationship for measuring cell viability. The right graph presents absorbance measurements across various PHB concentrations (0% to 10%) over different incubation periods. Across all concentrations, absorbance increased up to 72 hours, suggesting cell proliferation, followed by a slight decline at 96 hours. Statistical analysis (one-way ANOVA with Tukey’s post hoc test) indicated no significant differences (ns) in cell viability between PHB concentrations, demonstrating that PHB exhibited no cytotoxic effects at concentrations up to 10%.

# 3. RNA Sequencing (RNA-seq) Analysis

## 3.1. Sample Preparation

![](https://static.igem.wiki/teams/5404/res-fg6.png)

[Figure 6: Image of the samples for the RNA-seq. Duplicate samples of Synechocystis grown at pH 7.5 and 11 were used for the RNA-seq analysis (A total of four samples]

* **Selection of Strains:** Duplicate samples of Synechocystis grown in pH 7.5 and pH 11 were used for the RNA-seq analysis (Total four samples).
* **RNA Extraction:** Extracted total RNA using Trizol.

## 3.2. Transcriptome Sequencing

* **Library Construction:** Prepared Illumina TruSeq RNA library for RNA-seq.
* **Sequencing Platform:** Utilized Illumina TruSeq RNA technology for high-throughput sequencing.

![](https://static.igem.wiki/teams/5404/res-fg7.png)

[Figure 7: Overview of the general process to prepare a Illumina TruSeq RNA library for sequencing.]

Total RNA is first isolated from the sample of interest, such as cells or tissue. If DNA contamination is present, genomic DNA is removed using DNase. The RNA's integrity and total quantity are then assessed. Two main methods are available for library preparation: mRNA selection and rRNA depletion. Next, the purified RNA is fragmented into smaller pieces suitable for sequencing and reverse transcribed into cDNA. Sequencing adapters and sample-specific index sequences are attached to the ends of the cDNA fragments to allow the identification of individual samples in multiplexed sequencing. The resulting products are purified and amplified by PCR to create the final cDNA library. The library is then quantified and qualified to ensure readiness for sequencing. Typically, the insert size for RNA-Seq libraries ranges between 200-400 base pairs, and in paired-end sequencing, both ends of the fragment are sequenced to the desired read length.

![](https://static.igem.wiki/teams/5404/res-fg8.png)

[Figure 8: Overall general data analysis workflow]

After sequencing, the raw reads undergo quality control, with basic statistics such as overall read quality, total bases, and total reads calculated to ensure the data is suitable for analysis. If the sequencing cycle exceeds the insert size of the library, adapter sequences may appear at the 3' end of the reads. To reduce biases, these adapter sequences and low-quality bases are trimmed, and the trimmed data's basic statistics are recalculated. For transcriptome resequencing, the trimmed reads are aligned to the reference genome using HISAT2, a splice-aware aligner. Gene and transcript-level expression profiling is quantified with StringTie in reference-guided mode, considering strand-specific libraries when necessary. The expression profiling matrix provides read counts and normalized values like FPKM and TPM, which account for transcript length and coverage depth. Differentially expressed genes (DEGs) are identified by statistical testing between comparison groups, filtered by fold change and p-value criteria. Gene-set enrichment analysis is performed on significant gene lists to gain biological insights using Gene Ontology and KEGG pathway databases to identify relevant biological functions and pathways.

## 3.3. Differential Gene Expression Analysis

![](https://static.igem.wiki/teams/5404/res-fg9.png)

[Figure 9: Hierarchical Clustering Heatmap of expression levels for all genes analyzed in RNA-seq analysis.]

The heatmap displays the results of hierarchical clustering analysis using the Euclidean distance metric and complete linkage method, clustering genes and samples based on expression levels from the significant gene list (filtered by |fold change| ≥ 2 and exactTest raw p-value < 0.05). The heatmap shows that the duplicate samples from pH 11 and pH 7.5 exhibit highly similar gene expression patterns within their respective conditions, indicating minimal variation between replicates. This confirms that the samples were correctly prepared and processed, ensuring the reliability of the RNA-seq data for subsequent analyses.

![](https://static.igem.wiki/teams/5404/res-fg10.png)

[Figure 10: Volcano plot showing DEGs between the initial and adapted strains. The log2 fold change and p-value obtained from comparing the average of each group, plotted as a volcano plot. (X-axis: log2 fold change, Y-axis: -log10 p-value]

The volcano plot illustrates the log2 fold change versus -log10 p-value obtained from comparing the average gene expression levels between the initial (pH 7.5) and adapted (pH 11) Synechocystis strains. The x-axis represents the log2 fold change, indicating the magnitude of gene expression differences, while the y-axis represents the -log10 p-value, reflecting the statistical significance of those differences. Blue dots represent the 67 genes that are significantly underexpressed in the adapted strains, while yellow dots represent the 40 significantly overexpressed genes. This visualization highlights the key changes in gene expression as a result of adaptation to the high pH environment.

![](https://static.igem.wiki/teams/5404/res-fg11-1-new.png)

![](https://static.igem.wiki/teams/5404/res-fg11-2.png)

![](https://static.igem.wiki/teams/5404/res-fg11-3.png)

![](https://static.igem.wiki/teams/5404/res-fg11-4.png)

![](https://static.igem.wiki/teams/5404/res-fg11-5.png)

[Figure 11: List of differentially expressed genes (DEGs) identified through RNA-seq analysis of Synechocystis sp. grown under pH 11 conditions compared to pH 7.5. Genes highlighted in red are overexpressed, while those in blue are underexpressed. The fold change (pH11\_5/pH7) and corresponding p-value are provided to indicate the magnitude of expression difference and statistical significance.]

# 4. Discussion

## 4.1. Enhanced CO₂ Capture and PHB Production

* **High pH Environment:** In our experiment, PHB content peaked at pH 11.0 with a PHB content of 31.3 wt%, a significant increase compared to a PHB content of 20.0 wt% observed at pH 7.5. This is due to facilitated efficient CO₂ capture as bicarbonate ions, increasing availability for photosynthesis and PHB synthesis. However, higher pH conditions past the pH threshold may lead to decreased PHB content, most likely due to disruptions in cellular metabolism caused by extreme pH stress. As a result, optimizing pH at 11 is the best in facilitating PHB synthesis and CO₂ production.
* **Synergistic Effect:** Adaptation to the high pH environment not only improved tolerance but also enhanced metabolic pathways involved in PHB production. This may be due to shifts in resource allocation or rerouting of metabolic pathways in response to increased CO₂ availability and pH stress. However, the exact mechanisms underlying these changes remain unclear and warrant further investigation.

## 4.2 Key Upregulated Genes with Potential Relevance to PHB Synthesis:

The upregulation of genes related to sugar metabolism (mannose, transketolase), energy storage (polyphosphate kinase), and other metabolic pathways suggests that Synechocystis may be rerouting its carbon and energy resources toward PHB synthesis in response to the high pH environment. The hypothetical proteins and sensory kinases may also play a role in this regulation, but their exact functions would need further study.

1. **Alkaline Phosphatase:** This enzyme could be involved in providing phosphate groups necessary for cellular processes. Increased phosphate availability may impact the synthesis of key intermediates in the PHB production pathway.
2. **Mannose-1-phosphate Guanyltransferase:** Mannose metabolism is associated with nucleotide sugars like GDP-mannose, which could link to carbohydrate metabolism. Sugars and sugar phosphates are essential in generating acetyl-CoA, the precursor for PHB synthesis.
3. **Transketolase:** This enzyme is a part of the pentose phosphate pathway, which produces NADPH and precursors for biosynthesis. Increased NADPH production is essential for PHB synthesis, as it provides reducing power for the biosynthesis of fatty acids that lead to PHB accumulation.
4. **Polyphosphate Kinase:** This enzyme is involved in polyphosphate metabolism, which could influence cellular energy storage. An increased energy pool might favor the diversion of carbon into storage polymers like PHB.
5. **GDP-D-Mannose Dehydratase:** This enzyme could participate in carbohydrate metabolism by modifying sugar nucleotides. This could affect carbon flux towards acetyl-CoA, which feeds into PHB biosynthesis.
6. **Alpha-Mannosidase:** This enzyme, involved in carbohydrate degradation, may help regulate the flow of sugar substrates, potentially influencing PHB synthesis by balancing carbon flow toward storage polymers.

### Hypothetical Proteins:

Many of the hypothetical proteins might play a role in either sensing the high pH environment or directly or indirectly regulating metabolic pathways related to PHB synthesis, but further functional characterization would be required to clarify their roles.

### Hybrid Sensory Kinase:

This kinase could be involved in environmental sensing and might help the cells adapt to the high pH environment, possibly triggering pathways that lead to increased storage polymer PHB as a stress response.

## 4.3 Key Downregulated Genes and Potential Impact on PHB Synthesis

The downregulation of genes related to photosynthesis, protein synthesis, stress response, and carbon metabolism may indicate a general shift in cellular energy and resource allocation. The cell might be conserving energy and redirecting carbon toward the production of PHB as an energy and carbon storage mechanism under the stress of high pH conditions. This metabolic rerouting likely helps Synechocystis survive in unfavorable environments by building up storage compounds like PHB, which can later be metabolized when conditions improve.

1. **tRNAs and Ribosomal Proteins (tRNA-Ser, tRNA-Val, tRNA-Gln, 16S Ribosomal RNA, 50S Ribosomal Protein L16, 30S Ribosomal Protein S1, Elongation Factor TS, Elongation Factor EF-G, Peptidyl-tRNA Hydrolase):**
   * **Role:** These genes are involved in protein synthesis and translation machinery.
   * **Impact:** A decrease in translation and protein synthesis may suggest that the cell is conserving energy and resources, possibly redirecting carbon and energy toward storage molecules like PHB instead of producing new proteins.
2. **Phycobilisome Proteins (Phycobilisome LCM Core-Membrane Linker Polypeptide, Phycobilisome Rod-Core Linker Polypeptide CpcG, Allophycocyanin a Chain, Phycocyanin b Subunit, Phycocyanin-Associated Linker Protein):**
   * **Role:** These are involved in light harvesting in photosynthesis.
   * **Impact:** Reduced activity in light-harvesting complexes may indicate a shift in energy use away from photosynthesis. In Synechocystis, this could reroute cellular energy and carbon from photosynthetic processes toward the biosynthesis of storage compounds like PHB, especially under stress.
3. **Photosystem Proteins (Photosystem I Subunit II, Photosystem II PsbT Protein, Photosystem II Manganese-Stabilizing Polypeptide, Photosystem I Subunit III):**
   * **Role:** Involved in photosynthetic electron transport and energy production.
   * **Impact:** Downregulation of these components may limit photosynthetic capacity. In a high-pH environment, cells may reduce photosynthesis and focus on survival strategies, including the accumulation of energy storage molecules like PHB.
4. **Oxidative Phosphorylation and Carbon Metabolism (OxPPCycle Protein, Carbonic Anhydrase, Inorganic Pyrophosphatase, N-Acetyl-Gamma-Glutamyl-Phosphate Reductase):**
   * **Role:** These proteins are involved in energy metabolism and carbon assimilation.
   * **Impact:** Reduced carbon fixation and energy production through oxidative phosphorylation may conserve resources for alternative carbon storage, including PHB synthesis.
5. **Stress-Response Proteins (Rehydrin, OmpR Subfamily, Heat Shock Protein GrpE, DnaK Protein):**
   * **Role:** These are stress response proteins that help the cell manage environmental stress.
   * **Impact:** Lower expression of stress-response proteins may indicate that the cells are adapting to the pH stress by entering a storage mode rather than a defense mode, conserving resources and enhancing PHB production as a protective mechanism.
6. **Carbon Storage and Fatty Acid Biosynthesis (Gamma-Glutamyl Phosphate Reductase, Biotin Carboxyl Carrier Protein of Acetyl-CoA Carboxylase):**
   * **Role:** Acetyl-CoA carboxylase is crucial for fatty acid synthesis, which is a precursor for PHB synthesis.
   * **Impact:** Interestingly, downregulation of acetyl-CoA carboxylase subunits might seem counterintuitive, but it could indicate a regulatory shift where fatty acid synthesis is redirected or slowed, allowing more carbon flux into PHB synthesis.
7. **ABC Transporters and Other Regulatory Proteins (ABC1-Like Protein, Ferric Uptake Regulation Protein, Negative Aliphatic Amidase Regulator, PxcA):**
   * **Role:** These proteins are involved in nutrient transport and regulation.
   * **Impact:** Reduced nutrient uptake (e.g., iron via ferric uptake regulation) could trigger a shift in metabolism, favoring internal resource storage. PHB, being a carbon and energy storage polymer, could be synthesized as a response to decreased availability of essential nutrients.
8. **Hypothetical Proteins:**
   * While their exact functions are unknown, they may play a role in the regulation of metabolic pathways, environmental stress response, or cellular adaptation mechanisms.

## 4.4 Key Downregulated Genes and Potential Impact on PHB Synthesis

![](https://static.igem.wiki/teams/5404/res-fg12.png)

[Figure 12: Gene-Enrichment and Functional Annotation Analysis using KEGG pathway identifies four significant pathways in both downregulated and upregulated genes.]

Next, we performed KEGG pathway analysis to identify which pathways were involved in the upregulated and downregulated DEGs. The KEGG Pathway database (http://www.kegg.jp/kegg/pathway.html) integrates various types of omics data, including molecular information (e.g., genome sequences, structures), chemical information (e.g., metabolism of glycans and lipids), and molecular interaction data (e.g., physical interactions and co-expression). An enrichment test based on KEGG Pathways was conducted using the significant gene list. The results indicated that downregulated genes were primarily associated with pathways such as aminoacyl-tRNA biosynthesis, photosynthesis-antenna proteins, and photosynthesis. In contrast, only one pathway was associated with upregulated genes: fructose and mannose metabolism.

# 5. Conclusion

Our results support the modified hypothesis that culturing Synechocystis sp. under high pH conditions selects strains with enhanced PHB production. However, when the pH exceeds a critical threshold, PHB synthesis and bacterial growth cease. The adaptive laboratory evolution (ALE) approach successfully developed strains capable of thriving in alkaline environments while efficiently converting atmospheric CO₂ into valuable bioplastics, PHB. Gene enrichment and functional annotation analysis using KEGG pathways were consistent with the key genes identified in earlier sections of the study. Notably, photosynthesis-related genes were significantly downregulated, suggesting that high pH levels impair the photosynthetic machinery of Synechocystis.

Unexpectedly, we found that fructose and mannose metabolism pathways were upregulated in high pH conditions. This upregulation likely plays a key role in enhancing PHB production, as fructose and mannose metabolism increase the flux of carbon intermediates, particularly acetyl-CoA, which is a direct precursor for PHB synthesis. Additionally, the heightened activity of these metabolic pathways could provide excess reducing power (NADH, NADPH), which is essential for driving the biosynthesis of PHB. This metabolic shift towards carbohydrate processing, while photosynthesis is suppressed, allows the cells to channel carbon and energy towards PHB production as a stress response to the alkaline environment. Overall, these findings highlight a metabolic reprogramming in Synechocystis sp. that favors bioplastic production under alkaline stress, offering insights into optimizing PHB biosynthesis in cyanobacteria.

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