• Overview

Faced with the current serious plastic pollution problem, we have chosen to contribute by improving the production of degradable plastics through a three-layer dynamic regulation.

• Current issues with plastic

Because of its advantages such as durability, flexibility, and affordability, plastic has become an important part of our lives. Traditional petroleum-based plastics typically include polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), polyvinyl chloride (PVC), low density polyethylene (LDPE), or high-density polyethylene (HDPE), which are accumulate in environment at a very high rate and are harmful to our human being (Fig.1).

The School of Life Science of Shandong University is located in Qingdao, Shandong Province, which is closed to the sea. Thus, we have many opportunities to go to the seaside for internship and investigation. We found that plastic pollution is not a topic that far away from us. When not cleaned up in time, large amounts of plastic can accumulate on the beach.

Plastic pollution is all around us, but we wanted to understand the problem from a more comprehensive perspective, so we decided to conduct a series of preliminary human practices.

Meanwhile, it is important to note that plastics are derived from fossil fuels. The World Economic Forum has reported that 4%—8% of annual worldwide oil consumption is now connected to plastics. If we continue to depend on plastics, it is projected that they will contribute to 20% of all global oil consumption by 2050.

First, we collaborated with the "Life Sandbar" team at Shandong University to organize beach cleaning activities. This not only improves our environment, but also gives us a clearer understanding of the plastic waste situation around us.

And, we worked with other iGEM teams in various regions to collect soil samples and detected microplastic pollution in various regions through a series of experiments. At the same time, with permission, we conducted questionnaires, interviews and visits to get a deeper understanding of plastic pollution. Then,we conducted correlation analysis to find the inner link.

• Biodegradable plastic and PHB

Biodegradable plastics have gained widespread attention in recent years, among which bacterial polyhydroxyalkanoates (PHAs) have shown great potential. PHAs are biodegradable thermoplastics, and they are produced by bacterial as a kind of intracellular carbon storage compounds (Fig.3).

PHB (poly-β-hydroxybutyrate) is a class of PHAs. PHB possesses better physical properties than polypropylene for applications such as food packaging and is completely nontoxic.

We found that PHB product can be used in extensive aspects, ranging from medical industrial to household items (Fig.4).

After understanding the disadvantages of traditional plastics, we decided to choose degradable plastics as the general direction of our project.

Through the investigation of the types, properties and production costs of biodegradable plastics, we found that the commercial production of PHB, as a good biodegradable plastic, is greatly limited.

One reason is that the PHB production pathway conflicts with the central metabolic pathway. Another reason is that PHB is an intracellular product, which cannot be released automatically and needs to be artificially lysed. So, we decide turn to these two problems.

• Focusing on two problems

We are going to addressing these two specific problems using different tools:

• Problem 1: The PHB production pathway conflicts with the central metabolic pathway.

• Problem 2: As an intracellular product, PHB cannot be released on its own.

• Esa I/R system

The Esa I/R system is quite special from traditional QS system. The EsaI/R QS system is homologous to the LuxI/R QS system and originates the maize pathogen--Pantoea stewartii subsp. stewartia. EsaR can act as both transcriptional activator and repressor.

P_esaR is a natural EsaR-repressed promoter, whereas P_esaS is anatural EsaR-activated promoter. At low cell density (low ρ), EsaR binds to its esa box to turn off PesaR and turn on P_esaS. In the presence of AHL, EsaR can bind to AHL and release from the DNA. Thus, at high cell density(high ρ), the P_esaR is turned on and the P_esaS is turned off.

• Autolysis system of phage

We will use the lambda phage lysis gene cassette to design our auto-lysis system.

• Three-layer dynamic regulation model

All in all, we design a three-layer dynamic regulation model to solve our problems:

In the first and second layer, we using a QS-switch to regulate the flow of acetyl-coA. At the early stage of growth, using QS-switch turn on the TCA cycle and turn off the PHB production pathway, so that acetyl-coA flowed into the TCA cycle and the cells grew. When the cell grows to a certain extent, the TCA cycle is turned off, while the PHB production pathway is turned on, and the acetyl-coA flows to the PHB production pathway for PHB production.

In the third layer, we decide to use a stationary phase promoter and an auto-lysis system. The stationary phase promoter is used to regulate the expressing time of the downstream gene.When the bacterial reach the stationary phase, the promoter will turn on and then the cell lysis.

We obtained ten valid results from the experimental construction as follows

An example: we can see from it that its three stages successfully achieved separation.

We applied our model to PHB production and found that the extracellular PHB content of the model with the three-layer dynamic regulation model was much higher than that of the one without the complete three-layer dynamic regulation model.

• Further development