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 and arise safety concerns. Therefore, We implemented rigorous safety measures, covering from the kill switch in our engineering bacteria, the sterilization in the assembly of hardware, and laboratory safety facilities.

Kill Switch Design

Doc-Phd Toxin-Antitoxin System

For the final version of the kill switch, we decided to use an induced circuit. We had initially considered using an unnatural amino-acid-based kill switch, but we had to abandon that idea due to financial and time constraints. Additionally, we have a future plan to design a temperature-controlled kill switch, which is detailed in the temperature-sensitive kill switch section.

The inducer of our kill switch is IPTG. With IPTG present, the bacteria can survive, but if it leaks into the environment without it, the bacteria will die.

We initially decided to use the Doc-ssrA tag compound with TEV protease. When IPTG is present, it blocks the lacI operator, which in turn blocks the Plac promoter. However, this double inhibition actually enhances the process, allowing the downstream cI to be expressed. This then blocks the pCI promoter and turns off the expression of TEV protease(BBa_K627008). The CI component is a transcription repressor taken from E. coli phage lambda, which previous teams have used, and the code for this basic part can be seen at BBa_C0051.

The toxin compound, made up of the Doc toxin and SSrA tag, is connected by the TEV site (BBa_K3392000) and is usually produced by the powerful T7 promoter. The compound will not harm the bacteria if IPTG is present and no TEV is produced. However, suppose there's no IPTG in the environment. In that case, the TEV protease will be expressed and identify the seven-amino-acid TEV site and divide the peptide between the residues Q and G. This results in the death of the bacteria. The SSrA tag decreases the likelihood of leaky expression of the Doc toxin, with endogenous proteases ClpAP and ClpXP42 degrading the Doc linked with it.

During the process of constructing the plasmid, we discovered the antidote to the Doc toxin, namely Phd(BBa_K3143002). As a result, the genetic circuit can be further simplified in a more straightforward and convenient manner. The J23119 promoter was utilized to facilitate the expression of the Doc gene, while the Plac promoter was employed to regulate the expression of the Phd gene. The outcomes obtained were consistent, whereby the presence of IPTG in the system led to the expression of the Phd gene, resulting in its combination with the toxin. Conversely, in the absence of IPTG, the bacteria were released, leading to the activation of the Doc gene and subsequent bacterial cell death.

Doc-Phd toxin-antidote system

Unnatural Amino Acid Auxotrophy

Since we mostly contain our engineered bacteria in the hardware, we reckon that utilizing substance constraints is feasible. Therefore, we use a toxin-antidote system relying on an unnatural amino acid, 3-iodo-L-tyrosine(IY). Only when fed with IY can the engineered bacteria stay alive, which can be added directly to the hardware. If the bacteria unexpectedly escape or leak, it cannot survive without IY, leading to automatic death.

This is achieved using an amber stop codon and an amber-specific acyl-tRNA synthase. 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 Colincin E3 by forming a complex with it. When provided with IY and the expression of Full IY-tRNA synthase and amber suppressor tRNA, bacteria could incorporate 3-iodo-L-tyrosine(IY) into the antidote. Therefore, the engineered bacteria can stay alive only with foreign supplements of IY.

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

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.

Schematic demonstration of unnatural amino acid auxotrophy

Temperature-Mediated Switch

The inducer for our kill switch is IPTG, a substance that enables bacterial survival. If IPTG is not present and the bacteria escape into the environment, they will not survive.

Here's how the genetic circuit works: IPTG blocks the lacI operator, which prevents the Plac promoter from functioning, similar to how it operates in the lac operon. This dual inhibition enhances the process, allowing downstream Phd expression, which then combines with the Doc toxin, inactivating it to make the bacteria survive.

We also proposed a future plan to create a temperature-regulated kill switch. For this design, we want to make it work as a tempearture sensitive part, working in a relatively high temperature and restoring in a relatively low temperature in the facility. We set the working temperature as 37℃ and restoring temperature as 25℃, when the temperature is out of this range, the kill switch will be turned on and kill the engineering bacteria. Here's how we designed the part and how it works.

We chose Pr promoter, a strong promoter of E.coli λ phage regulating the early transcription, and to regulate this promoter we chose CI⁸⁵⁷, a temperature-sensitive mutant of repressor CI. CI⁸⁵⁷ can form a dimeric to bind the Pr promoter at 30℃ and thus the promoter is blocked, downstream gene transcrption repressed. It can release at 30℃ so that the promoter can work again, allowing the downstream gene to be transcribed.

There is a temperature-sensitive RBS downstream, which can work at a high temperature, and when the temperature is 25℃ to 37℃, the transcript will form a blocked status according to the princeple of complementary base pairing. Free energy stays at low level constantly with a little rise as the temperature ascends and the secondery structure stays steady as a stem-loop. When the temperature is above 37℃ the blocked structure is open and the mRNA can be translated.

We chose relBE to form a toxin-antitoxin system. The toxin, RelE, is capable of cleaving mRNA in the ribosomal A site cotranslationally, while the antitoxin, RelB, binds and inhibits RelE, and regulates transcription through operator binding and conditional cooperativity controlled by RelE. The temperature-sensitive RBS controls the RelB, following a normal RBS and RelE. We add a protein tag in the downstream of the RelB. The linker is TEV site with the seven amino acid sequence: E-N-L-Y-F-Q-G. TEV protease is able to recognize this sequece and cleave the peptite between the Q and G. The linker connects the RelB with the interaction domain of the RelE(no DNA binding domain).

Temperature-mediated kill switch

The kill switch works as follows:

At the restoring temperature(25℃), the CI⁸⁵⁷ binds the Pr promoter, and thus the toxin-antitoxin system doesn't work.

At the working temperature(37℃), the CI⁸⁵⁷ restriction is released, and both RelB and RelE can be transcribed and express antitoxin and toxin，TEV protease cleaves the tag so that RelB is able to bind RelE, and neutralization the system. As a result, E.coli functions normally.

Sequence of TA system

Sequence Alignment of RelE and RelB(Bøggild, A., 2012)

When the bacteria leak from the machine, as is known that the environmant temperature is always below 25℃, so the temperature-sensitive RBS can't work, with no RelB produced, so the RelE can killl the bacteria. In case the external temperature is high( above 37℃), we designed the tag. TEV protease loses activity quickly as the tempearture exceeds 37℃, so it can't cleave the linker, and RelB will bind the closest RelE interaction domain(i.e., the tag), being not available to neutralize the toxin, and bacteria die.

Hardware Safety

In addition, our hardware adhered to a predetermined and standard operating method for the workflow. This not only reduced the likelihood of any potential safety issues occurring, but it also lowered the risk of any engineered bacteria escaping into the environment. Hardware page has other details pertaining the NOX kit that may be found.

Lab Safety

General Safety

All research conducted by UCAS-China 2023 is in accordance with the 2023 iGEM safety policies and rules.[1]&[2] All of the parts used and/or generated by us are on the iGEM 2023 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.

Safety Basics, including the First aid box, storage cabinet, reagent storage room and the eye wash and sharps container

Reference

1. Miladi, B., Bouallagui, H., Dridi, C., El Marjou, A., Boeuf, G., Di Martino, P., Dufour, F., & Elm'Selmi, A. (2011). A new tagged-TEV protease: construction, optimisation of production, purification and test activity. Protein expression and purification, 75(1), 75–82.
2. Bøggild, A., Sofos, N., Andersen, K. R., Feddersen, A., Easter, A. D., Passmore, L. A., & Brodersen, D. E. (2012). The crystal structure of the intact E. coli RelBE toxin-antitoxin complex provides the structural basis for conditional cooperativity. Structure (London, England : 1993), 20(10), 1641–1648.
3. Wang, X., Han, J. N., Zhang, X., Ma, Y. Y., Lin, Y., Wang, H., Li, D. J., Zheng, T. R., Wu, F. Q., Ye, J. W., & Chen, G. Q. (2021). Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli. Nature communications, 12(1), 1411.
4. Hoynes-O'Connor, A., Hinman, K., Kirchner, L., & Moon, T. S. (2015). De novo design of heat-repressible RNA thermosensors in E. coli. Nucleic acids research, 43(12), 6166–6179.
5. Zadeh, J. N., Steenberg, C. D., Bois, J. S., Wolfe, B. R., Pierce, M. B., Khan, A. R., Dirks, R. M., & Pierce, N. A. (2011). NUPACK: Analysis and design of nucleic acid systems. Journal of computational chemistry, 32(1), 170–173.
6. Gazit, E. & Sauer, R. T. (1999). The Doc Toxin and Phd Antidote Proteins of the Bacteriophage P1 Plasmid Addiction System Form a Heterotrimeric Complex. Journal of biological chemistry, 274 (24), s. 16813–16818.