Safety

While processing the experiment of cyanobacteria does provide a number of benefits to our project, we are very cautious about the safety of our experiment since the negative impacts of cyanobacteria and experimental materials can be a disruption to the ecosystem and the environment. Therefore, to avoid all deleterious consequences, our team used professional safety measures to prevent accidents.

We used both E. coli and cyanobacteria as chassis. For E. coli, we utilize DH5α and BL21(DE3). They are classified as being in biosafety level 1. As for cyanobacteria, we use Synechococcus elongatus PCC 7942, which presents low or nonharmful effect to humans and the environment.

All the parts we used, including IsPETase and BhrPETase, are well-documented in numerous academic papers and are widely utilized in research. The most significant modification we made was introducing minor missense mutations—changing just one codon in each sequence, which affects only one amino acid—to enhance the efficiency of the existing enzymes. This process would produce our target enzyme without producing any toxins or byproducts.

As an iGEM team, the main part of our project is to insert modified genes into microorganism chassis’s like E. coli and cyanobacteria. Thus, there’s a high risk of manufactured products leaking out to the environment, which will cause severe and irreversible environmental issues. It is essential for us to develop a suicidal system for our microorganisms to guarantee that none of our products can survive when leaked out.

Our choice of chassis, Synechococcus elongatus PCC 7942, is a freshwater algae that cannot survive in saltwater, which ensures safety in the marine bioreactor. In addition, inspired by last year's project, we introduced an iron-dependent toxin-antitoxin suicide system into Synechococcus elongatus PCC 7942.

We try to use the two different toxin-antitoxin pairs (sepA1/sepT1 (Pemk-like system) and sepA2/sepT2 (Vapclike system)) with two different plasmids. Antitoxins are expressed using a light-inducible promoter PpsbA2 from Synechocystis sp. PCC 6803 which will be active in cyanobacteria under normal cultivation conditions, while toxins are expressed using the Fe3+ ion repressed promoter PisiA7942 from Synechococcus elongatus PCC 7942 respectively.

After 14 days of incubation, we measured the OD750 of BS3 (PpsbA2-sepA1-PisiA7942-sepT1), BS4 (PpsbA2-sepA2-PisiA7942-sepT2) to represent the growth of cyanobacteria.

Before our team began the experiment, our advisors provided training on lab safety and the proper use of lab equipment. Everyone learned the correct procedures for operating the devices and was reminded of their potential hazards. Additionally, we returned each piece of equipment to its designated place after use. This step is crucial, as it helps others and prevents items from getting lost or obstructing others. For every lab we conduct, at least one instructor supervises us.

Inside the laboratory, we separated a specific area for nucleic acid dye and electrophoresis to avoid contamination.

To enhance our protection, we are equipped with PPE such as lab coats, safety goggles, and gloves.

In order to prevent accidents, each of us learned how to use emergency equipments installed in the labatory. For example, if a fire acts, an acid leaks, or if we breathe in harmful gas, we all know how to react.

In the lab, we check the cables and functionality of electrical devices before use, wear protective gear, and properly label and store all items. These steps help reduce the risk of accidents.