iGEM 2025 — Team mingdao

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{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
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## What Should this Page Contain?

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* Record all activities in chronological order, with each entry clearly dated.
* Provide sufficient detail for each activity, including:

+ Purpose of the activity.
+ Methods and procedures used.
+ Results obtained.
+ Observations and notes.

* Document all experiments, protocols, data analysis, meetings, brainstorming sessions, and other project-related activities.
* Include photos, videos, and diagrams to illustrate your progress.
* Indicate which team members participated in each activity.

## Inspirations

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* [2024 EPFL](https://2024.igem.wiki/epfl/notebook)
* [2024 JU-Krakow](https://2024.igem.wiki/ju-krakow/notebook)

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{% block title %}Engineering Success{% endblock %}
{% block lead %}The ups and downs of science experiments{% endblock %}
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![Image of a landfill]({{static_asset_img}}/engineering-insert.webp)

## The Current Problem

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From your day-to-day coffee cups to take-out containers, polylactic acid, or commonly known as PLA, has become the face of “green” plastics. It promises biodegradability as an eco-friendly alternative to other petroleum-based plastics. Yet, beneath this sustainable image lies a delightful scheme. Currently, PLA is classified under the recycling code number #7 (Other), a category that contains all the "other" miscellaneous plastics that are rarely recycled in practice. Even worse, despite its eco-friendly reputation, PLA doesn't even readily decompose in the natural environment. Natural bacteria and enzymes struggle to degrade its tough and high molecular weight structure; only under elevated temperature and a controlled conditions environment of specialized composting equipment will it break down. Those so-called “biodegradable” plastics are no different from our conventional plastics. As a result, much of the PLA nowadays ends up in landfills or the environment, as it's too expensive and complicated for companies to bother recycling, allowing it to persist in our environment.

PLA is a polyester made up of repeating units of lactic acid linked by ester bonds. Its molecular structure is highly crystalline and hydrophobic, which makes it resistant to breakdown by water and natural microbial enzymes. Unlike organic matter such as fruits or plants, PLA does not easily provide access points for microbial attack, meaning soil bacteria and common enzymes struggle to break it down efficiently.

When bacteria and enzymes capable of degrading PLA are present, the process remains slow and inefficient. High-molecular-weight PLA requires specialized enzymes, such as PLA depolymerases, to hydrolyze its ester bonds. Even then, most of these reactions occur only under elevated temperatures or in controlled composting conditions. At ambient temperatures found in soil or water, degradation is extremely limited. Furthermore, when lactic acid monomers accumulate outside bacterial cells during degradation, they can lower the pH of the environment and inhibit further enzymatic activity. This creates a feedback effect that slows down the process even more.

As a result, this year, our goal is to promote the replacement of petroleum-based plastics with PLA in Taiwan. We began by investigating how PLA is classified and degraded. Sooner or later, in the course of studying the recycling and degradation process of PLA, we found that although several ester bond hydrolysis enzymes have already been commercialized, none of them are specifically designed for PLA degradation. This observation indicates that there remains considerable potential to further optimize PLA-degrading activity. Therefore, we aimed to enhance the degradation efficiency of enzymes toward PLA through mutagenesis by introducing extensive mutations into existing enzymes and employing auto-induction for rapid screening. In the end, we successfully identified variants with significantly improved degradation efficiency compared to the wild type, thus improving the efficiency of the degradation. Follow-up will be the process of our designing and testing process, and how we manage to mutate and improve our enzyme.

## Cycle One

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#### Design: Finding PLA-degradable enzymes and testing their activities

The mechanism of PLA-degrading enzymes involves the hydrolysis of ester bonds in PLA by enzymes such as proteases, lipases, cutinases, and esterases. This process generates soluble products (lactic acid monomers and oligomers) and partially degraded, insoluble PLA residues, which are analyzed by different biochemical and physical methods (Shalem A, Yehezkeli O, Fishman A. Enzymatic degradation of polylactic acid (PLA). Appl Microbiol Biotechnol. 2024 Jul 10;108(1):413. doi: 10.1007/s00253-024-13212-4. PMID: 38985324; PMCID: PMC11236915.)

![Figure 1]({{static_asset_img}}/engineering-fig1-new.webp)

Figure 1 Credit: Appl Microbiol Biotechnol. 2024 Jul 10;108(1):413.

To investigate the workflow and degradation efficiency of available enzymes, we first considered the source of three commercially available proteases and lipases, whose enzymatic activities have been well characterized. In addition, we found an enzyme that has higher specificity to PLA. We regarded these four genes as the wild type, and we conducted assays on them to collect data on their efficiency of enzymatic PLA degradation.

#### Build: gene cloning

The genes of Tritirachium album Proteinase K (PK), Bacillus licheniformis Alcalase (AC), Candida antarctica Lipase B (CA), and Amycolatopsis sp. PLA Depolymerase (DP) were synthesized by Integrated DNA Technologies, Inc., with fragment sizes of 1107 bp, 884 bp, 951 bp, and 671 bp, respectively. First, the synthesized DNA fragments were used as templates for PCR to amplify the target genes, as shown in Figure 2 below.

![Figure 2]({{static_asset_img}}/engineering-fig2.webp)

Figure 2

The gene fragments were cloned into pET-28a for high-level protein expression under IPTG control, driven by the T7 promoter. Following ligation and transformation into E. coli DH5α, the plasmids were extracted and verified by PCR using T7 promoter (T7P) and terminator (T7T) primers, as well as by restriction digestion with EcoRI and PstI, as shown in Figure 3.

![Figure 3]({{static_asset_img}}/engineering-fig3.webp)

Figure 3

#### Test: pNPB and Lactate production with PLA degradation assay

The pET-28a vectors carrying AC, CA, DP or PK were re-transformed into E. coli BL21 for protein induction along with the empty vector as a negative control for assays and GFP/pET-28a as a positive control for gene expression. First, we picked a single colony for each kind of vector in the 6 ml culture of LB broth supplemented with 50 μg/ml of kanamycin. The overnight cultures were 100x diluted to 300 ml of fresh medium. When the OD600 value reached 0.6-0.8, 0.3 mM of IPTG was added for protein induction at 20°C, 180 rpm for 36 hr. The bacterial cultures were washed and centrifuged, then subjected to sonication using Qsonica Q125 Sonicator (Amplitude 40%, Time 15 min, Pulse 15 sec on and 30 sec off). The lysates were collected and filtered through 0.45 μm PES membrane as crude enzyme extracts for functional assays.

pNPB is a fast and reliable measurement for ester bond hydrolysis, which is used to evaluate esterase or lipase activity. For quick understanding of our enzyme activities, the enzymes were subjected to pNPB assay with 1 mM pNPB in 100μl of 20 mM Tris buffer at pH 8 for 10 min at 37°C. The color change to yellow means the hydrolysis of the ester bond of pNPB, which can be measured at OD420 nm, indicating the release of p-nitrophenol and thus enzyme hydrolytic activity.

To examine the activity of PLA degradation, the enzyme lysates (46 μl) were incubated with PLA powder (0.01 g) at 50°C for 30 min. After centrifugation, the lactate in the supernatants was analyzed by the L-Lactate Assay Kit (Sigma-Aldrich). Lactate production was measured at OD587. The results are shown in Figure 4-7.

![Figure 4]({{static_asset_img}}/engineering-fig4.webp)

Figure 4

![Figure 5]({{static_asset_img}}/engineering-fig5.webp)

Figure 5

![Figure 6]({{static_asset_img}}/engineering-fig6.webp)

Figure 6

![Figure 7]({{static_asset_img}}/engineering-fig7.webp)

Figure 7

As shown in the figures, PLA Depolymerase and Proteinase K exhibited higher enzyme activity in pNPB assay, resulting in greater lactate production from PLA degradation. However, lactate production still faces challenges for practical applications. In contrast, in pNPB assay, Alcalase and Candida antarctica Lipase B exhibited lower enzymatic activity and the reduced PLA degradation efficiency compared to the control groups, including the GFP control and vector-only control.

#### Learn

We found that the currently available enzymes for PLA degradation in the market are not optimized for PLA. Even the specialized Poly(L-Lactic Acid) Depolymerase from the Amycolatopsis or Paenibacillus strain, which has higher specificity to PLA, still does not optimally degrade certain domains within the PLA structure under specific conditions. Therefore, this project aimed to optimize these enzymes by mutation.

## Cycle Two

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#### Design

This study aimed to generate a large library of mutant enzyme-expressing variants and employ the method of auto-induction to screen potential variants. The gene cloning strategy described above was used to express the mutant enzymes. Following this protocol, we then assessed whether mutagenesis improved the efficiency of enzymatic degradation.

#### Build

We have found eight approaches to random mutagenesis in the paper of Bitesize Bio.

Throughout the study, we carried out GeneMorph II EZClone Domain Mutagenesis Kit for producing the targeted gene’s mutant variants, so we can delve more into the versatility of a protein. First, we selected the targeted genes as the model, and we adopted the Mutazyme II DNA polymerase to run the error-prone PCR. The part of mutant megaprimer synthesis that amplifies the gene fragment and purifies the targeted fragment with mutations.

This is introduced in the paper as a technique for driving amino acid substitutions in proteins, domains, or promoters by mutation introduced into PCR. (GeneMorph II EZClone Domain Mutagenesis Kit Instruction Manual Catalog #200552 Revision E.0)

Soon, the mutated PCR product would serve as the megaprimer and be annealed to the original donor plasmid to run the EzClone reaction. With the specialized enzyme mix is a 2x formation alongside its high-fidelity DNA polymerase to extend the whole plasmid backbone and insert the mutant into the targeted sites, the completeness of other plasmid sites can be maintained. This can prevent the unwanted secondary mutations to occur in the cloning process. To eliminate the parental DNA template, we added the Dpn I restriction enzyme, specific for methylated and hemimethylated DNA, into the reaction to digest the parental DNA template. That means the final product remains only the new plasmid of the random mutagenesis.

As it is carried out, we would transform the mutant plasmid to the competent cell E.coli DH5α to culture. Then into another competent cell, E.coli BL21. Each colony on the agar plate represents an independent mutant variant. All colonies gather and become a complete mutant library as we proceed with the colony pickup.

However, in cycle 1-3(test) , we found that the LB+IPTG system could not meet the needs of high-throughput measurement. Therefore, we later followed our developed measurement system(For details, please see Measurement) and used the ZYM-5052 auto-induction medium to express the protein. The essence of auto-induction medium is the adoption of the mixture of the carbon substrates. (Mehrnoosh Fathi-Roudsari et al.) It initially began with the glucose to provide fasten growth, lactose for inducing the expression, and glycerol for providing the sustained energy source. This design of the gene expression stage can automatically accomplish the induction, and there is no need to add in the IPTG inducer, which would result differently in the variants.

The lab workflow started by letting the E.coli strain with plasmid inoculated into the LB broth supplemented with the kanamycin antibiotic. The protocol then proceeds into culturing the cell by setting the temperature 37 degrees celsius and spinning in the incubator with 200 rpm for hours. As the cell approached the log phase, they were subcultured at a 1:100 ratio into the ZYM-5052 medium, and we proceeded culturing them. In the beginning, glucose served as their primary source to support fast proliferation, and lactose as an inducer has limited functions because of the absence of cAMP/CAP. When the glucose is fully depleted, lactose will start entering the cell and transfer into allolactose to forbid the Lacl from inhibiting. This contributes to the expression of the T7 RNA polymerase, thereby initiating the transcription and synthesis of the target protein. Likewise, the glycerol continues to provide a sustained energy source, so the induction remains progressive. After 16 to 20 hours, proteins accumulate extensively in the host cells. The culture can only be centrifuged at a low temperature in order to harvest the cells and proceed to the purification.

By following this flow, the culture growth, induction, and expression can all run smoothly. Compared to the induction by IPTG, this approach enables us to achieve a high-level production of proteins at high cell densities. It also increases the stability and efficiency of the lab experiment. For more details, please refer to the measurement webpage.

#### Test

We followed the same approach mentioned above to run the pNPB assay and the lactate production with the PLA degradation assay. The results are in Figure 8-11.

![Figure 8]({{static_asset_img}}/engineering-fig8.webp)

Figure 8 -pNPB of Alcalase(AC) wild type and mutations

![Figure 9]({{static_asset_img}}/engineering-fig9.webp)

Figure 9 -pNPB of Candida antarctica Lipase B (CA) wild type and mutations

![Figure 10]({{static_asset_img}}/engineering-fig10.webp)

Figure 10 -pNPB of PLA Depolymerase (DP) wild type and mutations

![Figure 11]({{static_asset_img}}/engineering-fig11.webp)

Figure 11 -pNPB of Proteinase K (PK) wild type and mutations

The results of the pNPB assay showed that after comparing the mutant variants to the GFP and the wild type; we selected a few mutant variants with greater enzymatic activity. These include A6, A13, A14, C5, C6, C11, D3, D13, D16, P2, P7, P15 to test on the lactate production for the PLA degradation assay, and the vector only and wild type served as the control groups in this assay.

![Figure 12]({{static_asset_img}}/engineering-fig12.webp)

Figure 12 -Lactate production assay

#### Learn

We found that the Proteinase K’s mutant variant (P2 group) has the best degradation efficiency via the data collected by screening. It produced 15 times more lactate than the vector-only control group. This explains that the lactate we’ve tested was produced from PLA, not the metabolism of the host strain. Moreover, the amount of lactate produced is at least 1.6 times higher than the PK wild-type control group. This further justifies that random mutagenesis enhances the ability of the genes to degrade PLA. Although the PLA degrading enzymes in the lab exhibited significant activities on PLA degradation, the condition still needs to be optimized before implementing the application on real-life target fields such as the organic farms. In the future, we hope a work may be carried out to present another way of degrading PLA degradation in real life. The details are presented in the future-work section.

## References

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1. GeneMorph II EZClone Domain Mutagenesis Kit Instruction Manual Catalog #200552 Revision E.0.
2. IslandPubDev518. “Random Mutagenesis: 8 Approaches for Success.” Bitesize Bio, 12 Dec. 2007, bitesizebio.com/252/8-approaches-to-random-mutagenesis/?utm\_source=chatgpt.com%29%EF%BC%9B%E6%88%91%E4%B8%8D%E7%9F%A5%E9%81%93%E6%88%91%E5%80%91%E7%94%A8%E7%9A%84%E6%98%AF%E5%93%AA%E5%80%8B. Accessed 14 Sept. 2025.
3. Mehrnoosh Fathi-Roudsari, et al. “Auto-Induction for High Level Production of Biologically Active Reteplase in Escherichia Coli.” Protein Expression and Purification, vol. 151, 8 June 2018, pp. 18–22, www.sciencedirect.com/science/article/abs/pii/S1046592818301815#:~:text=Auto%2Dinduction%20media%20are%20made,catabolic%20repressor%20of%20lac%20promoter., https://doi.org/10.1016/j.pep.2018.05.008. Accessed 14 Sept. 2025.
4. Studier, F William. “Protein Production by Auto-Induction in High-Density Shaking Cultures.” Protein Expression and Purification, vol. 41, no. 1, 8 Feb. 2005, pp. 207–234, pubmed.ncbi.nlm.nih.gov/15915565/, https://doi.org/10.1016/j.pep.2005.01.016. Accessed 19 Sept. 2025.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
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## What Should this Page Contain?

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Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

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* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
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## What Should this Page Contain?

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* Clearly and objectively describe the results of your experiments and research.
* Use tables, graphs, and images to visualize your data.
* Discuss the implications of your findings in the context of your project goals.
* Highlight any trends, patterns, or significant observations.
* Include an analysis summary section to synthesize your findings and discuss their overall significance.
* Outline your future plans for the project, including potential applications, further research, and development.
* Explain what went wrong and what you learned from these experiences.
* Discuss potential improvements or alternative approaches.
* Emphasize the importance of scientific honesty and transparency.

## Inspirations

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* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 EPFL](https://2024.igem.wiki/epfl/results)
* [2024 UToronto](https://2024.igem.wiki/utoronto/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 Sydney-Australia](https://2024.igem.wiki/sydney-australia/results)
* [2024 Toulouse-INSA-UPS](https://2024.igem.wiki/toulouse-insa-ups/results)
* [2024 BNDS-China](https://2024.igem.wiki/bnds-china/results)
* [2024 Vilnius-Lithuania](https://2024.igem.wiki/vilnius-lithuania/results)
* [2024 XMU-China](https://2024.igem.wiki/xmu-china/results)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/results)

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