Safety has always been our top priority. In the project, we are redesigning artificial systems and reconstitute biological elements, utilizing materials that may be dangerous. All of which may pose threat towards natural ecology and surrounding environment thus arise safety concerns. Therefore, effective safety measures are applied, such as inserting a kill switch in the engineering bacteria and laboratory safety facilities.

Unnatural Amino Acid Auxotrophy

To prevent the engineered bacteria escaping into surroundings and causing damage to the environment, we use a toxin-antidote system relying on an unnatural amino acid(Uaa), 3-iodo-L-tyrosine(IY). Only when fed with IY can the engineered bacteria stay alive, which can be added directly to the medium. If the bacteria unexpectedly escape or leak, it cannot survive without IY, leading to automatic death. The bacteria in our project are contained in the lab in most of the situation, hence this Uaa killing switch is an effective and feasible way to prevent the leakage.

This is achieved using an amber-specific acyl-tRNA synthase and an amber stop codon. Colicin E3(colE3) is a highly toxic RNase that kills the host bacterium with a few molecules. 1amb-IMME3 could directly inhibit the activity of Colicin E3 by forming a ColE3-ImmE3 complex. In the ON-state, IY is provided, with the expression of Full IY-tRNA synthase and amber suppressor tRNA, bacteria could incorporate 3-iodo-L-tyrosine(IY) into the antidote, In contrast, IY is absent in the OFF-state, translation of the target gene products is terminated at the UAG, resulting in no functional proteins being produced, and the bacteria will die of toxicity. The amber stop codon is inserted next to the AUG translational start codon of immE3(1a-immE3) to prevent the production of truncated protein. Therefore, the engineered bacteria can stay alive only with foreign supplements of IY. Since IY is a synthetic molecule and does not exist either in the natural environment or in living organisms, an unexpected turning on of the switch by an environmental or an endogenous Uaa is impossible.[1] A relative study revealed that the kill rate could be rapid and reliable, with the escape frequency of 71 thousand cell divisions-1 escaper. The killing half-life is estimated to be 49.2+-7.2min, and the viability of bacteria could be reduced quickly upon removal of IY.[2]

To further optimize the killing switch, quadruple-TAG-inserted IYRS with a Met 6 Val substitution [4a-IYRS(M6V)] is involved. 4 constitutive UAG stop codons prevent the leakage translation attributable to the basal read-through in the OFF-state. Since the Met 6 may be an alternative translation initiation site and may cause leaky expression of IYRS, we import a M6V mutation. A research showed that the net gain, defined as yield in the ON-state/leakage in the OFF -state, of 1a-IYRS(M6V) is 27-fold higher than that of the unmodified IYRS.[3]

Though there could still be leakage detected in the absence of IY, it could be manipulated by controlling the concentration and the optimization of the expression density and balance of aaRS/tRNA.

Lab Safety

General Safety

All research conducted by UCAS-China 2024 is in accordance with the 2024 iGEM safety policies and rules. All of the parts used and/or generated by us are on the iGEM 2024 White List. Throughout the whole team of UCAS, all national and institutional rules and regulations are strictly followed.

Staff Safety

All of the team members had to follow multiple introduction sessions given by the lab manager. These introductions elaborately covered safety policies and rules inside the specific labs. Every freshman entering the lab must read the manual on Laboratory Safety and Hazardous Chemicals Safety. Besides, anyone who is permitted to use our lab must pass the laboratory safety and skill exam at 90% correctness or above.

Storage and Environmental Safety

Our lab has equipped many facilities that keep our experimental environment safe. The location of all safety equipment was shown during this introduction. All the specific hazards are locked in a hazardous warehouse which can only be opened by two lab managers, detailed in the Safety Form.

Different wastes, such as general waste, biohazards and sharps, should be deposited into separate bins. Smoking, eating, and sleeping are strictly prohibited during the experiment. After the experiment, every staff should maintain cleanliness and orderliness in the laboratory, and clean up waste materials following the waste disposal protocols.

Reference

1. Kato Y. (2019). Translational Control using an Expanded Genetic Code. International journal of molecular sciences, 20(4), 887.
2. Kato Y. (2015). An engineered bacterium auxotrophic for an unnatural amino acid: a novel biological containment system. PeerJ, 3, e1247.
3. Kato Y. (2018). Tight Translational Control Using Site-Specific Unnatural Amino Acid Incorporation with Positive Feedback Gene Circuits. ACS synthetic biology, 7(8), 1956-1963.