Abstract

Plastic mulch residue has caused environmental problems, such as depleting the organic matter in the soil or releasing of greenhouse gas. Our project aims to propose a solution from the perspective of biodegradation. In our project, we introduce alkane 1-monooxygenase gene(alkB) from Rhodococcus into Streptomyces to test whether it will be endowed with capability of degradation of low molecular weight polyethylene (LMWPE). Since Streptomyces are one of the most abundant Actinobacteria in soil environment , we asses that the successful transfer of the alkB gene will greatly enhance the environmental self-degradation capability of plastic debris.

Beyond the experiment, our project also incorporates community engagement efforts to maximize its impact. We surely hope the scientific knowledge in the project can benefit more people.

Introduction

Since 1950th, the invention of plastic mulching has changed agriculture. In coming decades, plastic mulching has become a widely used technique due to their multifunctional benefits, including regulation of surface temperature and humidity, suppression of weed growth, and enhancement of crop quality.

However, every rose has its thorn. The residue of mulch exhibits non-biodegradability in soil. This factor can potentially cause negative effects. For instance, the residue can change the permeability of soil, causing the reduction of moisture in soil. A large accumulation of residues may also lead to the release of toxic substances.

In fact, the impact caused by the residue of plastic mulch may be measured by centuries. The main composition of mulch is polyethylene, which is extremely undegradable in natural environments. 10-year laboratory experiments showed that LDPE buried in soil reduced its weight by only 0.2% per year, and Long-term (32–37 years) studies by Ohtake et al. (1998) estimated a period of approximately 300 years required for complete degradation of a 60 μm thin LDPE layer.

Our team focuses on finding the potential bio-degradation solution to this problem. Meanwhile, we notice that If people have a clear understanding of the harm posed by residue, the pollution problem caused by people's indifference can be greatly resolved.

Thus, trying to find solutions at the social layer is also an important consideration in our experiment.

Wet Part

The specific biodegradation method is based on a gene fragment. alkB, alkane 1-monooxygenase gene, is able to degrade n-alkanes. alkB is widely distributed in Rhodococcus, Pseudomonas aeruginosa, Bacillus subtilis. Research on this gene is usually associated with petroleum degradation work. In our research, alkB gene fragment is imported into Streptococcus to test if it could gain ability to degrade LMWPE(the main composition of residue), while analyzing the enzyme derived from the expression of alkB.

Firstly, why alkB & Streptococcus are chosen? The alkB gene, as its name suggests, is highly correlated with the degradation of short-chain hydrocarbons. However, given the structure of low molecular weight polyethylene and the operating mode of the enzyme AlkB expressed by the alkB gene (which targets carbon chains for carboxylation), alkB has certain potential in the research on the decomposition of low molecular weight polyethylene. Hyun Jeong Jeon has already imported alkB gene from Pseudomonas to E-coli. and the research indicates the E-coli successfully degrade LMWPE. Inspired by this and considering the high abundance of Streptomyces in the soil, we introduced the alkB gene into Streptomyces tk24 to test its heterologous expression effects and its degradation capability for LMWPE. The modified strain's ability to degrade LMWPE suggests that introducing this strain into the environment may enhance the environment's self-degradation capacity.

Secondly, How we do cloning? We use traditional enzyme digestion and Golden Gate Assembly in our research. This can help us improve our efficiency and increase fault tolerance. More important, we want to compare the advantages and disadvantages between two methods. The enzyme used in traditional way is EcoRI and HindIII, the enzyme used in Golden Gate Assembly is BsaI. Two shuttle plasmid, pUWL201 and pSF1C-A-RFP are used as backbone.

Thirdly, How we do rest of work? We use a combination of conjugal transfer method to introduce plasmids into the target strain. Conjugation transfer utilizes E. coli as the donor strain. Finally, the screened strains are inoculated for LMWPE degradation assessment.

Dry Part

We also design and build learning models which improve the overall quality of our project. We first collect data indicating the situation about the use of plastic film different areas of China. Then, based on this data, we use random forest classifier to build a model which can evaluate whether using a specific plastic film is plausible. Meanwhile, we use AlphaFold not only to predict the protein expressed by our target sequence but also further construct a model which can help with optimizing the target sequence to make it more adaptive to host bacterium.

Mulch Advisor Our primary goal is to help people decide whether the plastic film they choose is reasonable. In this case, we collect different kinds of data which can help us achieve this goal. For instance, we pick different city at 4 regions of China, then collect data about the most used plastic mulch in a certain city. Besides, we also gather some related information such as weather conditions, soil composition, etc. Then, the data is used to train model which is based on the random forest classifier, and the test set is separated to verify the function of model. We also preserve option for entering mulch information by user himself, as a result, a single user can have his own result from model according to his own condition.

SeqOpt This pipeline begins with the idea of optimizing the coding sequence of a target enzyme across species. We load the wild-type gene, define protected regions, and build a scoring function with a fine-tuned protein language model. This is integrated into an MCMC framework that proposes point mutations and accepts or rejects them using the Metropolis criterion.

Human Practice

As we mentioned in introduction part, trying to find solutions at the social layer is also important. Even though the time is limited, we still plan to start a club in our high school to promote our project and conduct an investigation to understand and help people who truly use plastic mulch.

School Club We start a new club at our high school mainly for this project. We hope that more students and our classmates will gain interest in science and have awareness of protecting environment. We design and exhibit our poster during school festival, and also organize speech with many students. We hope the club could make more students not just focusing on doing tests.

Investigation This survey aims to understand how farmers in Henan Province use plastic mulch and how residual plastic film left in the soil affects crop yield. The research focuses on farmers’ usage habits, disposal methods, awareness of soil pollution, and attitudes toward biodegradable mulch. The survey was conducted both online and offline, targeting about 100 farmers from different regions of Henan, including Nanyang, Sanmenxia, Zhumadian, Zhoukou, and Shangqiu. To ensure clarity and accessibility, most questions were designed as multiple-choice items, covering five main topics: basic information, mulch use, perceptions of residual film, awareness of biodegradable mulch, and open suggestions.

Reference

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