All of our team members passed a Laboratory Safety course provided by Aalto University before entering the lab facilities. The training covered the following topics:
- Lab access and exits
- Emergency situations
- First-aid practices
- Recycling and waste handling
- Chemical safety
We worked with Biosafety level 1 organisms, namely Escherichia coli (TOP10 & C2566) and Saccharomyces cerevisiae (W303α). Being Biosafety level 1, these E. coli and S. cerevisiae strains were safe and non-pathogenic.
We understand the potential risks related to release of Genetically Modified Organisms (GMOs) into the environment and we have adhered to iGEM’s official Do not release policy concerning GMOs. Proper handling and disposal protocols were followed while working with GMOs. Utmost care was taken to prevent contamination of the environment of any sorts by following standard laboratory procedures and sanitation. Everyone working in the wet lab had proper training and personal protective equipment.
COVID-19 regulations were adhered to while working in the laboratory at all times by maintaining social distance, wearing face masks and periodic hand sanitization.
GutLux was made using GMOs which were modified by plasmids that encode human proteins, hence we have put in place some efficient and fool-proof mechanisms that ensure biological safety.
Since GutLux is an ingestible biosensor and the organisms involved are genetically modified, a kill switch is included in the design of the capsule to prevent leakage of the GMOs both inside and outside the subject’s body. The design for the kill switch was from our partner team – Thessaly. The proposed kill switch makes use of a biological AND gate system adopted from a similar AND gate system published in 2007 (Anderson et al., 2007), to avoid leakage of a bacteria outside of the body.
The components of the AND gate in our kill switch are designed to ensure that our engineered cells can only survive in the hypoxic conditions of the gut. The two inputs of the AND gate system for E. coli are:
- The SupD gene that encodes for a modified tRNA which recognizes amber stop codons (UAG) and add a serine instead of stopping the translation. The SupD gene is under a hypoxia-inducible promoter, PfnrS .
- A gene encoding a repressor protein which is expressed under a constitutive promoter. The gene itself is mutated to contain amber stop codons where normally serine would be present in at least 3 positions.
An equivalent AND gate will also be designed for S. cerevisiae, by using yeast specific components.
The output of this AND gate is based on the production of a toxin. When both the promoters are active (i.e., both inputs work), the repressor protein will be expressed. This will block the production of a toxin which can kill the cell. So, when the capsule is outside the gut, SupD cannot be expressed and consequently, the repressor will not be expressed as well. This sequence of events will result in the production of a toxin that kills the cell.
By using the same system, leakage of GMOs inside the gut can also be prevented by incorporating a molecule that inhibits the repressor. This inhibitor will be housed in a different chamber of the capsule. In the case of unlikely breakage of the capsule inside the gut, the repressor will come in contact with this inhibitor and get inactivated. Thus, the toxin will be produced and the cells will get killed.
Another potential safety concern that required addressing was potential leakage of plasmids encoding human proteins inside the gut which might lead to unintended expression outside the capsule. To tackle this, we have conceptualized a tRNA based expression control system inspired from the Biotechnology and Synthetic Biology chapter in Brock biology of microorganisms (pages 395-396, 15th edition) (Madigan et al., 2019). The system utilizes the same tRNA system of Amber stop codon (UAG) from the AND gate that can add serine instead of stopping the translation. The coding sequences contained in the plasmids can be mutated to include amber stop codons in place of serine codons. This way it will be very unlikely, even with the risk of horizontal gene transfer, that the coding sequences contained in the plasmid can be correctly expressed anywhere else than inside our engineered cells.
These biological safety mechanisms are theoretical as of the writing of this wiki and have been thoroughly researched to fit our needs. Proper testing of the safety mechanisms and their subsequent optimization would have been possible had time been on our side.
We acknowledge the existing regulations for processing personal and medical data in the design of our wireless data transmittance. The data collected will only consist of concentration values and will not include any personal identifiers, such as age, gender or sexuality. Collecting personal data from the patient is up to the researcher. To map any safety risks and seek out general concerns about data transmittance safety issues, we conducted a Data Privacy and Security workshop in collaboration with iGEM Team Thessaly. Details and outcomes of the event can be found in Partnership.
Based on the workshop, we designed our research tool so that it will only measure concentration values, which are transmitted with radio frequency to the receiver. The receiver has a local memory that is wiped after the information is downloaded to the researcher’s computer. This process enables patient anonymity, since we retain collecting any additional personal information.
Risk of retention or physiological damage has been considered at each step of our capsule design. The clinical safety has been taken into account when designing each of the capsule components. The most important safety considerations were capsule shape and dimensions, material choices, electric component toxicity and signal transmittance. The component choices are discussed in detail on our Hardware page. In addition, we researched EU medical device regulations and consulted a company that provided in-depth knowledge on regulations and quality management of medical devices. You can read more about the clinical safety research and applying regulations under Implementation.
1. Anderson, J. C., Voigt, C. A., & Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Molecular systems biology, 3, 133. https://doi.org/10.1038/msb4100173
2. Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., Stahl, D. A., & Brock, T. D. (2019). Brock biology of microorganisms. NY, NY : Pearson.