Workspace
We were able to develop and conduct the majority of our experimental activities in the following laboratories:
Laboratory of Genetic Engineering, Centre for Biotechnology Studies, Universitas Gadjah Mada. Most of our work with EcN was conducted here.
Integrated Agrocomplex Laboratory, Faculty of Agriculture, Universitas Gadjah Mada. Due to fully booked lab equipment in the Laboratory of Genetic Engineering, Centre for Biotechnology Studies, our PI assigned us to this laboratory in late September.
Integrated Research Laboratory, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada. Our work with cancer cell lines was conducted here since there is a dedicated room for mammalian cell culture.
All of our labs are Biosafety Level (BSL) 2, equipped with open benches and Laminar Air Flow (LAF) hoods. Additionally, the lab technicians in each lab provided us with a brief introduction to lab safety rules and direct training on using certain tools for our experiments.
Strain Safety
We utilized Escherichia coli Nissle 1917 (EcN) as a chassis, which is non-pathogenic and commonly used as a probiotic [1]. Additionally, we used the epithelial colorectal adenocarcinoma cell line WiDr, which requires Biosafety Level 1 precautions for handling [2]. However, wearing a safety lab coat, gloves, masks, and frequently disinfecting with 70% alcohol were mandatory for us during the entire experiment. Detoxification of all tools and samples before (using an autoclave) and after use or contact with specimens (using 10% bleach incubation or boiling water) was also included on our safety lab checklist to prevent any contamination, leakage, and accidental strain release into the environment.
CRISPR-Cas Safety
The plasmids used in this project, pFREE and pCryptDel4.8 contain Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas-based genome editing tools targeting the endogen plasmid pMUT1 and pMUT2 respectively in our plasmid curing protocol [3]. Although in the end we didn’t successfully conduct the curing step, there are still concerns about alteration in pathogenicity, antibiotic resistance, and transmission of the bacteria. Thus, there needs to be comprehensive testing for our final product license [4].
Chemical Safety
We used Methylthiazolyldiphenyl-tetrazolium bromide, or MTT, to measure the cytotoxic activity of the modified EcN. This compound is known to cause skin and eye irritation and is also suspected of causing genetic defects [4]. To handle it safely, we ensured to check the Safety Data Sheet (SDS) before working and received training from the lab technician. We always wore gloves, masks, and lab coats while handling it. We disposed of all chemicals that came into contact with the cell line by using 10% bleach and incubating them overnight before disposal.
[1] Behrouzi A, Mazaheri H, Falsafi S, Tavassol ZH, Moshiri A, Siadat SD. Intestinal effect of the probiotic Escherichia coli strain Nissle 1917 and its OMV. Journal of Diabetes & Metabolic Disorders. 2020 Jun;19:597-604.
[2] WiDr; CCL-218; ATCC; December 4, 2022; https://www.atcc.org/products/ccl-218#detailed-product-information (accessed 2023-9-9).
[3] Kan A, Gelfat I, Emani S, Praveschotinunt P, Joshi NS. Plasmid vectors for in vivo selection-free use with the probiotic E. coli Nissle 1917. ACS synthetic biology. 2020 Dec 10;10(1):94-106.
[4] Rath J. Safety and security risks of CRISPR/Cas9. Ethics Dumping: Case Studies from North-South Research Collaborations. 2018:107-13.
[5] Methylthiazolyldiphenyl-tetrazolium bromide, ≦ 100%; CAS RN: 298-93-1; rev. 8.2; Sigma; September 14, 2023; https://www.sigmaaldrich.com/ID/en/sds/SIGMA/M2128 (accessed 2023-9-9).
