• The feasibility of the project

• Factors to consider when designing the safety module

• The best way to degrade microplastics

• If enzyme exocytosis strategy appropriate or innovative

In the early stages of our project, we gained an basic understanding of the global issue of microplastic pollution, especially the ability of mangroves, as a special barrier between marine and terrestrial ecosystems, to accumulate microplastics, which poses a serious threat to this ecological environment.

After we introduced our project and background and sought further feasibility of operation, Prof. Chen introduced the impact of microplastics on bacterial community diversity. He pointed out that microplastics not only affect microbial diversity but also alter important ecological functions in sediments, such as nitrogen cycling.

1. Safety Module

The suicide system for the engineered bacteria should consider the three areas of the ocean, mangroves, and land, restricting the engineered bacteria between the ocean and land. It is possible to simulate the leakage of microorganisms in the laboratory.

2. Experiment

In terms of the degradation method of microplastics by bacteria, Prof. Chen believed that enhancing the adsorption capacity of enzymes is more important than exocytosis, and that digestion by microorganisms after phagocytosis is more direct and efficient. He recommended to abandon the enzyme exocytosis strategy and focus on enhancing the degradation capability of microorganisms.

As for the details of execution of our experiment, It was suggested to collect soil samples from the Zhuhai Mangrove Forest for subsequent ecological and chemical analysis. We can visit Dr. Zhou who majors in the research.

(1) Direct Extraction: Extract PE directly from the soil samples or set up control and experimental groups for weight comparison.

(2) Bacterial Concentration: The concentration of the bacterial strain can be explored through a blank control plus different concentration gradients.

4. Engineering Bacterial QS System

It is suggested that this be deleted, as the engineered bacteria are difficult to survive and reproduce in the wild environment, and it is challenging to grow to the preset concentration, thus rendering the activation of the degradation enzyme system unlikely.

5. Pathway Design

Convert the fatty acid chains formed by the degradation of microplastics into an information flow rather than a material flow (such as hormones).

• How to develop the experiment given to the features of mangroves forests

• The most appropriate measuring methods

After being introduced by Prof. Chen, we made contact with Dr. Zhou Xu and asked him how to carry out a scientific research, especially how to conduct research and analysis on mangrove soil. Dr. Zhou made a detailed introduction and explanation on the following aspects:

1. Soil Sample Collection

To collect soil samples from the Zhuhai mangrove forest for subsequent ecological and chemical analysis, we have to sample by groups and multiple points (Randomly select 3 sampling points for each of the three tidal levels: high, medium, and low.), considering the random errors, precisely locating the quadrat, and clearly labeling them with "Sampling Point Number-Depth".

We should store samples at 4°C for refrigeration to maintain freshness.

2. Sample Processing

To analyze soil composition and understand the ecological and chemical characteristics of the mangrove soil, we'd better calculate C/N, C/P, and N/P ecological stoichiometric ratios; determine key components such as available phosphorus, available manganese, zinc, copper, nitrate nitrogen, organic carbon, and available iron.

Considering the need to maintain the ecological reduction of the soil, discuss whether to perform routine drying, picking, grinding, sieving, sorting, and bottling steps.

The experiment is to study growth preference of specific strains in mangrove soil and investigate its potential impact on soil ecology.

(1) Sterilization

Discuss whether to retain the original soil ecosystem, ultimately deciding to retain it.

(2) Soil Bottle Restoration

Place soil samples in plastic tubes in descending depth order.

Mark layers and maintain original slope to avoid disturbance.

(3) Strain Inoculation

Set up concentration gradients for strains.

(4) Cultivation and Simulated Tidal Conditions

Use sterile seawater to infiltrate soil every 12 hours for 2 hours.

Adjust infiltration level according to tide levels (high, medium, low).

Set up control plots with different infiltration durations.

(5) Sampling and Detection

Focus on sampling depth and adjust sampling intervals based on experimental needs.

4. Data Analysis & Modeling

(1) In an academic context, it is feasible to simulate tidal variations and construct predictive models.

(2) Use R language and Origin software to statistically analyze the data, create charts, and thoroughly analyze the experimental results.

5. Field study arrangements

We made a reservation with Dr. Zhou to carry out field sampling activities following Dr. Zhou's guidance into the Zhuhai Qi'ao Island Mangrove Nature Reserve, and we prepared the equipment, props, list and experimental design needed for sampling in advance.

6. Field practice learning of soil sampling

On July 5, 2024, We underwent field sampling training led by Dr. Zhou, in the Zhuhai Qi'ao Island Mangrove Nature Reserve, where we collected soil samples according to the plan and completed the sampling process. During our visit to the mangrove reserve, we also observed some plastic waste. After completing our sampling, we packed up and took away all the plastic waste we saw.

• How to design and construct useful plasmids

• Some specific questions and details about constructing plasmids

Prof. Chen furnished the following pivotal details and methods pertaining to the construction of plasmids:

1. Methods for Literature Retrieval.

2. We employed plasmids with low copy numbers due to their higher persistence in bacteria compared to high copy number plasmids. This reverses the commonly held notion that higher copy numbers are inherently better.

3. Search for Broad-Host-Range Plasmids.

After reviewing the meeting records, conducting extensive literature research, and reflecting on our approach, we selected the Broad-Host-Range Plasmid pAB1. The plasmid map, created with SnapGene, is shown below:

• If our concept of hardware feasible

Given the limited experimental time, we only need to demonstrate the connection of the biological detection module's protein, ensuring that a reaction can occur. If the reaction can occur, the protein structure will change and the bioelectric potential will definitely change, which can be captured as a signal. This means that our hardware design is feasible.

The Doctor suggested that to solve the issue of unstable water flow, we could first test the potential data from the blank background. After the data stabilizes, we can then add the sample for testing, which is equivalent to measuring the background first and then stripping it.

Dr. Wang provided suggestions on related hardware production, including information on manufacturers.

• • How to improve the experimental design for verification

Prof.Su pointed out that for mangrove ecosystems, which are at the interface of land and sea, the soil environment is greatly influenced by tides. In the actual soil verification, there are still significant differences between the soil in permanently flooded and intermittently flooded areas. This means that it is necessary for us to design multiple control experiments to simulate the degradation conditions under different soil environments.

Through this exchange, we further understood the impact of tides on the soil environment of mangroves and further improved the verification experimental design of the degradation module.

• • Inspiration for metabolic modeling

In the discussion, Dr. Ma mentioned the idea of using AI to predict intermediates in metabolic pathways, which provided important guidance and inspiration for our modeling work.

She suggested that machine learning and deep learning methods could be used to analyze the complex relationships within metabolic pathways, uncover potential metabolic routes, and thus predict the generation pathways of key intermediates. Dr. Ma's insights provided a new direction for our further research and laid a solid foundation for us to refine the methodology of metabolic modeling.

• • Suggestions on the projects in mangrove forest soil from experts

The original soil samples are not perfect replicates, with many variable factors (such as various other microorganisms, secreted substances, etc.). The professor believed that soil sterilization is better, which can make them replicates.

Spotting them together on a plate will definitely not merge, we can try cultivating them in the same liquid medium to see if they can coexist or if one side will dominate the other.

Try using artificial wetlands to replace mangrove soil for plastic film experiments. Experiments generally start from simple to complex, soil testing can be the last step, and it is suggested that we prioritize the cultivation experiment of experimental bacteria and plastic.

It is suggested to use statistical methods to see if the difference is significant.

In summary, the professor evaluated our experimental design as comprehensive and of significant importance, with a well-thought-out approach, such as using a citrate promoter that can recognize citrate concentration in the safety module.

• • Advice on overcoming the challenges brought by limited sample sizes

Specifically, Dr. Zeng explained in detail how to combine the prediction results of multiple models through ensemble learning to enhance the overall performance of the model. He also suggested that during the fine-tuning of the model, it is important to make full use of existing data for incremental learning to adapt to different application scenarios and data distributions. In addition, he emphasized the importance of data augmentation, especially when the number of samples is insufficient, by generating new training samples to expand the dataset, which can effectively improve the model's generalization ability.

Regarding the use of support vector machines, Professor Zeng specifically reminded us to pay attention to the handling of abnormal results. He suggested that in the final stage of the model, the prediction results should be strictly inspected and analyzed to identify and deal with possible outliers, thereby ensuring the reliability and stability of the model in practical applications.

In summary, these valuable suggestions not only helped us optimize the model but also prompted us to pay more attention to the quality of data and the verification of the model in subsequent research, laying a solid foundation for subsequent modeling.

• • If our detection works effective
• • Other ways of detection

During our fluorescence detection, we encountered discrepancies in test results between liquid and solid media, which posed confusion for our observations and inspections. After presenting our findings and consulting with Prof. Zhu, we gained a deeper understanding and insight into fluorescence detection, microbial detection, and other testing methods. This meeting significantly assisted us in validating our results and refining our procedural checks.

• • The effects of our model
• • How to use model to improve our metabolism system
• • What exact models should we focus on

Res. Zhang detailed the main directions and ideas of modeling, and pointed out that the problem of our current approach is that metabolic analysis fails to provide help for the verification of experimental results. He also offers the following advice:

1. Pay attention to whether the gene that expresses the enzyme introduced in the experiment really plays a role;

2. Through the simulation of changes in the content of products and important substances, we can truly reflect the purpose of the project and the effect it wants to achieve.

• • The effect of our tidal current dynamics model
• • How to improve the construction and narration of tidal models
• • How to analyze the influence of tidal current on flora more comprehensively

Dr. Li gave detailed answers to our questions about the tidal dynamics model and suggested that we could explore the influence of sediment by means of environmental sampling. From the perspective of sediment transport, the tidal process and sediment transport are related, but it may not be ideal to directly measure the samples and then explain the tidal dynamics.

At the same time, based on the shortage of tidal flow data, he also suggested that we use tidal data for alternative solutions. Because the sediment problem is very complex, the dynamics of different time scales can lead to large erosion and deposition, so the tidal analysis can be simplified to some extent a corresponding degree.

In addition, he helped us refine the language of our existing models.

• • Does our project work well?
• • How can we improve and finalize our project?

Prof. Adeel emphasized the need to enhance the project's presentation, feasibility, and communication. He suggested improvements such as clearer visuals and narratives, supplementary data, detailed methods, safety assessments, exploration of potential by-products, project renaming, and refined communication skills.

Here are the detailed suggestions from Prof. Adeel:

(1) Diagrams should be clearer, effectively showcasing the sources of microplastics and the bacterial action mechanism.
(2) The narrative should be improved by incorporating environmental issues, solutions, and the project's significance to increase its appeal.
(3) Clarify the project's value in addressing environmental issues and its contributions to sustainable development.

(1) Supplementary experimental data is needed to demonstrate the efficiency and feasibility of bacterial degradation of plastics and CO₂ fixation.
(2) Detailed descriptions of experimental methods and operational procedures are necessary to reflect the rigor of the project.
(3) Evaluate the bacterial safety to ensure no adverse effects on the microbial community in mangrove soil.

(1) Explore the potential use of Thalassia hemprichii seagrass as a by-product to enhance the project's economic feasibility.
(2) Consider renaming the project to more accurately reflect its core content.

(1) Strengthen the overall project presentation by using diagrams, narratives, and data to clearly convey its content.
(2) Improve the experimental design, supplement data, and conduct risk assessments to ensure project feasibility.
(3) Improve English writing and presentation skills to better communicate with international peers and showcase the project's value.