In 2019, Japanese officials cancelled a swimming event for the ITU Paratriathlon World Cup because of concerningly high levels of Escherichia coli (E. coli) in the water ^[1]. While most E. coli normally live in the intestines of humans and animals without causing any trouble, some strains of E. coli can cause sickness like diarrhea, contaminating water and food supplies ^[2]. Therefore, it is crucial to prevent E. coli contamination as much as possible.

One major area of concern is lab leakage of E. coli, made even more concerning by the fact that these E. coli are likely genetically modified. Being able to transfer DNA between themselves through conjugation, genetically modified E. coli can easily spread their genes to wild strains of E. coli, which has unknown, potentially harmful, risks ^[3].

Moreover, public concerns over biosafety pressure researchers to adopt measures against lab leaks even if there is no real threat ^[4]. Thus, a cheap and effective strategy for containing genetically modified organisms is needed.

Adhering to our mission of improving biosafety, our target customers are those with high possibilities of experiencing leaks, such as biochemical synthesis companies and high-level laboratories dealing with engineered bacteria. The significance of our product lies in its potential as a cheap alternative to conventional methods of biosafety and is widely compatibly with various experiments. Our product can mitigate the impacts caused by leakage. For instance, genetically altered bacteria can disrupt the genetic pool of environment outside the lab. With our product, the lab workers can safely dispose the bacteria. Since many labs already have their own safety protocols, our product can aid its efficiency and add a layer of safety. Furthermore, our product is not limited to E. coli, but it can be modified for other types of bacteria . The versatility of our product makes it more cost effective than those on the market.

A large scale bacterial leakage could, in itself, could cause dire consequences like disease, pollute the environment, and disrupt local ecosystem stability. Furthermore, other than the leaking of seemingly benign strains like E. coli, there have also been cases of stronger, more destructive strains being leaked from high-level laboratories.This shows the urgent need for the development of more effective and efficient E. coli treatment.

The damage, though, does not stop there. The leaking of engineered bacterium's genome also poses a threat to stability within natural environments. Genetic pollution, "the spread of genes in the environment, when they could not have got there by natural means^[5], poses great environmental and ecological threats. Genetically modified organisms when released into nature can out-compete native species, disrupt fragile ecosystems, and jeopardize biodiversity. Genetic pollution can lead to irreversible changes in the genetic makeup of native populations, potentially resulting in weakened resistance to diseases, reduced adaptability, and loss of unique traits. In a 2004 study, researchers observed that "the number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria ^[6]. Consequently, it disturbs the delicate balance within ecosystems and the ability of ecosystems to provide for life within. Currently, biochemical synthesis companies and high-level laboratories are the greatest customers for genetically modified microbes, which inherently makes them the most plausible source for leakages. Therefore, as our target customers, strictly enforcing biosafety measures is of paramount importance for biochemical synthesis companies to minimize the risk of genetic pollution. Stringent biosafety protocols and containment strategies are necessary to prevent accidental releases of genetically modified organisms into the environment, thereby minimizing ecological risks and preserving natural ecosystems and their invaluable contributions to our planet.

Accidental leakage of the genetically engineered bacteria could result in branches of confidential intellectual information. These bacteria containing the plasmids contain the work of various innovators, and if it's not disposed properly, the bacteria and their genetic material could fall into the hands of any external parties, threatening developers' intellectual properties.

It is therefore important to prevent the lab leaks and its damages. Current solutions to eliminating excess E. coli in the environment includes high-temperature sterilization, applying chemicals, introducing specific organisms, using UV light to disinfect, etc^[7]. These are either ineffective, as they might require specific environmental conditions to be carried out^[8], or they harm the environment as it indiscriminately kills other organisms^[9-10]. Innovative methods include constructing bacteria which requires a specific substance for growth or function, that the bacteria "starve" to death once it left the designated environment^[11-12]. However, these are all ineffective for their demand to be redesigned to fit different species or different experiments, costing extra time, energy, and resources.

Ultimately, we want to find a method that could prevent the environmental risk lab leaks pose and does so at high efficiency and low cost. The product therefore should be applicable to a wide variety of genetically engineered bacterium (or even generally applicable), reaching commercialization. To this end, we selected the external condition of temperature to trigger cell death, acting as a "kill-switch".

Our project's primary focus revolves around preventing contamination. Our design centers on enabling bacteria to undergo programmed cell death at lower temperatures, specifically around 22°C, while preserving their essential functions at higher temperatures, such as 37°C. This design ensures that in the event of a bacterial leak, the organisms will self-destruct when exposed to room temperature conditions, while still retaining their required functions at elevated temperatures^[13]. Using synthetic biology techniques, we developed a plasmid that triggers the death of host bacteria near 22°C, without interfering with their functions at temperatures of 37°C or above^[14-15]. This was achieved by placing a gene encoding a toxic protein under the control of a Lux operon, which governs the transcription of the toxic protein based on environmental temperature. Implementing this kill-switch mechanism only requires the transformation of our plasmid into the desired bacterial strain(Figure 1).

Figure1.The project design engineering drawing

With all that's said, our product is aimed at environmental problems caused by laboratory bacterial leaks and its harm to innovators' intellectual properties. Furthermore, we aim to create a product that is more cost-efficient and has greater efficacy than the previously listed alternatives. Therefore, we need to use synthetic biology techniques to construct a plasmid that functions as a "Kill-switches" that activates upon some external conditions. Temperature sensing systems should be used instead of the conventional nutrient-deficiency switch, to be able to fit different species without needing to drastically redesign the plasmid. To be more specific, we will utilize elements of the luxR/luxI type quorum sensing system to program a "kill-switch" that functions at room temperature—22 degree Celsius—and remain inactivated at the human body temperature—37 degree Celsius. By this, the leaked or disposed bacterium would undergo cell death when exposed to external conditions, providing a safety barrier for its potential damages.

Figure 2.The concept map of project objectives

[1] “[FALSE] Did Levels of E. coli Bacteria Exceed Acceptable Limits in the Swimming Area during the 2020 Tokyo Olympics Triathlon Events?” Hong Kong Baptist University (HKBU) Fact Check, 24 Aug. 2021, factcheck.hkbu.edu.hk/home/en/fc_report_eng/tokyo_olympics_triathlon/.

[2] “Questions and Answers | E. coli | CDC.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 1 Dec. 2014, www.cdc.gov/ecoli/general/index.html.

[3] Phornphisutthimas, Somkiat, et al. “Conjugation in Escherichia Coli.” IUBMB Journals, 13 Nov. 2007, doi.org/10.1002/bmb.113.

[4] Tompa, Rachel. “New Strategy Could Prevent GMO Leaks from Lab to the Wild.” Fred Hutch Cancer Center, 27 Jan. 2015, www.fredhutch.org/en/news/center-news/2015/01/preventing-gmo-leaks-from-lab-to-wild.html#:~:text=It’s unclear whether lab escapes are likely or,many researchers feel they should pursue containment strategies.

[5] Schell, Jeff. “Science and Agriculture in the 21st Century.” Developments in Plant Genetics and Breeding, 2000, pp. 17–25, https://doi.org/10.1016/s0168-7972(00)80094-8.

[6] Ishii, Nobuyoshi, et al. “Release of transforming plasmid DNA from actively growing genetically engineeredescherichia coli.” FEMS Microbiology Letters, vol. 240, no. 2, 1 Nov. 2004, pp. 151–154, https://doi.org/10.1016/j.femsle.2004.09.024.

[7] Kim N H , Cho T J , Rhee M S .Current Interventions for Controlling Pathogenic Escherichia coli[J].Advances in Applied Microbiology, 2017, 100:1.DOI:10.1016/bs.aambs.2017.02.001.

[8]Yu Z , Tan K , Quan W ,et al.Research on the application of ultraviolet disinfection technology in wastewater reuse and its running cost[J].Chinese Journal of Geochemistry ( English Edition ), 2006, 025(0z1):129.DOI:10.1007/bf02839987.

[9]Ciochetti, D. A., and R. H. Metcalf. 1984. Pasteurization of naturally contaminated water with solar energy. Appl. Environ. Microbiol. 47:223-228[Abstract/Free Full Text].

[10]Fayer, R. 1994. Effect of high temperature on infectivity of Cryptosporidium parvum oocysts in water. Appl. Environ. Microbiol. 60:2732-2735

[11]Lopez G , Anderson J C .Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21(DE3) Biosafety Strain.[J].Acs Synthetic Biology, 2015, 4(12):1279.DOI:10.1021/acssynbio.5b00085.

[12] Ryuichi Hirota, Kenji Abe, Zen-ichiro Katsuura, Reiji Noguchi, Shigeaki Moribe,

Kei Motomura. A novel biocontainment strategy makes bacterial growth and survival dependent on phosphate.Scientific Reports, 44748 (2017)

[13] Bazhenov, S.V., Scheglova, E.S., Utkina, A.A. et al. New temperature-switchable acyl homoserine lactone-regulated expression vector. Appl Microbiol Biotechnol 107, 807–818 (2023). https://doi.org/10.1007/s00253-022-12341-y

[14] Nocadello, S., Swennen, E.F. The new pLAI (lux regulon based auto-inducible) expression system for recombinant protein production in Escherichia coli. Microb Cell Fact 11, 3 (2012). https://doi.org/10.1186/1475-2859-11-3

[15] Hoffmann SA, Diggans J, Densmore D, Dai J, Knight T, Leproust E, Boeke JD, Wheeler N, Cai Y. Safety by design: Biosafety and biosecurity in the age of synthetic genomics. iScience. 2023 Feb 10;26(3):106165. doi: 10.1016/j.isci.2023.106165.