Overview

    Safety has always been the priority of NCKU_Tainan’s concerns. This year, to synthesize selenomelanin in vivo, various rules, techniques, and designs have been applied to improve the safety of the whole project, making the engineered bacteria more suitable for broader applications in sustainable space biomanufacturing. NCKU_Tainan believes that the advancement of synthetic biology comes from responsible acts.

Laboratory Safety

    NCKU_Tainan is in full compliance with the safety and security policies of iGEM competition.

    In the project, experiments were conducted in the laboratories of Prof. Masayuki Hashimoto, Prof. Han-Ching Wang, and Prof. I-Son Ng, which contain equipment for both Biosafety Level 1 and 2. After taking several lectures and an online examination, all wet members are credited with a "Basic Laboratories Safety Instruction" certificate from National Cheng Kung University. At the same time, two weeks of intensive training on equipment and chemical usage were also conducted, following protocols written by advisors. In addition, a supervisor or an instructor was always present in the labs.

Project Safety

Escherichia coli DH5α

    In the project, NCKU_Tainan used DH5α as a host in most experiments because it is one of the most commonly used wild-type E. coli strains and has no antibiotic resistance. Despite the little threat it may pose to the environment, NCKU_Tainan strictly follows laboratory safety procedures during the operation.

Hazardous Chemical

    Sodium selenite is one of the substrates for Sec synthesis; however, it contains toxicity towards humans if touched directly or inhaled. It also causes danger to the environment, especially for aquatic life. Therefore, instructions from the PIs were followed to comply with the safety rules of NCKU when using sodium selenite. Specifically, we put on gloves and glasses, performed experiments in a chemical fume hood, and discarded the waste appropriately according to the policies of laboratory waste liquid treatment.

UV Radiation

    UV-C and UV-B were utilized to confirm the function of selenomelanin. To avoid exposure to intense radiation, NCKU_Tainan always consulted experts before the experiment and never conducted it without professional help.

Environmental Safety

    In the project, NCKU_Tainan aims to engineer radiation-resistant E. coli by producing melanin and selenomelanin. However, this may increase the risk of bacteria leaking from laminar bench cabinets that use UV-C for sterilization. Being responsible iGEMers who value biosafety, it is necessary to design a kill switch not only for the project but also for the sustainability of the community.

Design

Fig. 1. The design of biosafety

    In selenomelanin production, extra Tyr was added into the medium as one of the precursors. This inspired us to knock out the tyrA gene, which is essential for Tyr synthesis in E. coli, so the bacteria can only take in Tyr from the medium and cannot live outside. However, the immediate depletion of Tyr for melanin synthesis may inhibit cell growth due to our kill switch design. Hence, we simulated the consumption curves at different Tyr concentration levels before constructing the kill switch. According to model results, there is no need to worry about running out of Tyr with the supply of at least 0.05 g/L of Tyr in the beginning (see Model page).

    The construction of the mutant strain includes three procedures[1]: PCR gene replacement method, P1 bacteriophage transduction and the deletion of the antibiotic resistance gene. To examine whether the kill switch functioned well, we cultured the strain with and without Tyr in the medium and measured the OD value. In Fig. 2, after 20 hours of culture, the bacteria cannot survive if not given additional Tyr, confirming the function of the designed kill switch in our engineered microbes.

Fig. 2. Examination of the function of tyrA

Device Safety

Microfluidic Chip

    The microfluidic chip was designed disposable to avoid any contamination or bacteria remaining inside the chip, which render our experiment results invalid. After every experiment, the chip was sanitized with an alcohol solution to kill the bacteria. It was discarded according to waste disposal regulations to avoid any leakage into the external environment.

MerStage

    In MerStage design, UV-C lamps were installed inside the device as a radiation source. Since UV-C cannot be seen with the naked eye, it might be harmful without noticing. NCKU_Tainan utilized 5mm-thick black acrylic to build up the walls of MerStage and block UV-C leakage. UV-C testing papers were used to ensure no radiation was transmitted through the wall.