Describe how and why you chose your iGEM project.
With the development of genetic engineering technology, the applications of genetically modified microorganisms (GMMs) become more and more widely, resulting increasing risks of GGM environmental leakage. However, the existing techniques cannot overcome their own set of limitations to prevent and control the GGM environmental leakage well, hindering their universal application. To address this, we propose to design a "multi-factor inducible self-destructive synthetic biology chassis" that not only caters to diverse production requirements but also maximizes biocontainment of GGMs, thereby offering an enhanced solution to this challenge.
Genetically modified microorganisms (GMMs) are defined as bacteria, fungi, or viruses in which the genetic material has been altered principally through recombinant DNA technology, in other words, by means that do not occur naturally (Stemke, 2004).
With the development of genetic engineering technology, the metabolic potentials of microorganisms are being explored and harnessed in various ways. So far, GMMs have been used in food industry, agriculture, medicine and health care, bioremediation, energy production, and other industrial biotechnology to improve chemical production to increase yields and reduce environmental impacts. Genetic engineering offers the advantages over traditional methods of increasing molecular diversity and improving chemical selectivity. In addition, genetic engineering offers sufficient supplies of desired products, cheaper product production, and safe handling of otherwise dangerous agents (Figure1, Lerner et al., 2024; Han, 2004).
As GMM technologies have become more refined and developed in increasing applications, society's concerns on the risks of GMM release have become more widespread. According to Stemke (2004), the environmental leakage of GGM will pose the following risks: (1) Horizontal DNA Transfer. One of the major safety concerns surrounding widespread use of GMMs is their ability to exchange DNA with other organisms in an uncontrolled environment. This horizontal gene transfer could potentially introduce new traits or properties into recipient organisms, further complicating the ecological and health implications of GMMs. (2) Environmental Impact. When GMMs are use in outdoor environment to remediate contaminated soils, improve soil fertility, manage pest control, and vaccinate livestock and wildlife, they may escape into the environment and disrupt natural ecosystems. This can occur through gene flow to wild organisms, leading to unintended consequences such as the spread of antibiotic resistance or the displacement of native species. (3) Human Risk. Apart from interactions with released GMMs in the environment or laboratory, as the increasing usage of GMMs or their associated DNA as whole GMM foods and probiotics and GMM vaccines, there are risks for humans: increased exposure to antibiotic resistance genes; which may result in transfer of antibiotic resistance genes to indigenous flora, transfer of genes accidentally or intentionally that might produce human pathogens; production of GMM toxins; and activation of human immune allergies.
To prevent and control the environmental release of GMMs, effective strategies and methods have been developed generally from physical control, chemical control and biological containment. Physical containment measures play a crucial role in preventing the release of GMMs into the environment. This includes the use of physical barriers such as biosafety cabinets and closed systems, which restrict the movement of GMMs. Additionally, autoclaving can effectively kill discarded GGM before dumping. Chemical control methods provide an added layer of protection against environmental release of GMMs. The use of disinfectants and sterilization agents, such as bleach or 75% alcohol, effectively eliminates any GMMs that may have inadvertently escaped containment. Biological containment strategies focus on engineering GMMs with genetic modifications that minimize their survival outside of controlled environments. Through the deliberate manipulation of their genetic makeup, scientists can restrict the ability of GMMs to replicate, survive, or establish themselves in natural environments. This can be achieved by introducing "kill switches" or genetic modifications that render the GMMs dependent on specific conditions that are only present within controlled environments.
(1) About Kill Switches A kill switch refers to a mechanism incorporated into GMMs that allows the researchers to exert control over their survival and reproduction. Typically, a kill switch consists of two core components, the killer system and the inducer system (Fudan, 2020). The killer system comprises essential genetic elements responsible for the activation of the kill switch, while the inducer system serves as an external trigger to activate the circuit. Usually the kill switch system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat (Figure 2, Moe-Behrens et al., 2013). The functionality of a kill switch lies in its ability to ensure the containment and prevention of GMMs from escaping into the environment. By controlling the reproduction and survival of GMMs, kill switches offer a reliable safety measure for engineered microorganisms. In the event of a potential leakage or unintended release, the kill switch can be triggered to induce programmed cell death, rendering the GMMs non-viable.
(2) Types and Characteristics of Commonly Used Kill Switches There are several types of kill switches commonly employed in engineering GMMs, each with its unique characteristics and applications. One such type is the repressible kill switch, which relies on the suppression of essential genes or pathways vital for GMMs' survival. By incorporating repressor proteins that can be artificially controlled, researchers can toggle the kill switch ON or OFF, thereby regulating the survival and containment of GMMs. This type of kill switch offers simplicity in design and is well-established in the field (Bondy-Denomy et al., 2013). Another prominent type is the toxin-antitoxin system, which involves the introduction of an artificial toxin and a corresponding antitoxin into GMMs. Under normal conditions, the antitoxin neutralizes the toxin, allowing the GMMs to thrive. However, upon activation of the kill switch, the antitoxin is degraded, thereby releasing the toxin and inducing programmed cell death in GMMs. This kill switch design is advantageous due to its reversibility and feasibility of fine-tuning toxin expression levels (Seed et Dennis, 2020).
(3) Limitations of Existing Kill Switch Technologies and Potential Improvements Despite the progress made in kill switch technologies, several limitations still need to be addressed to enhance their efficacy. Some kill switches may not be foolproof, such as the risk of mutation affecting the switch’s ability to work or premature/late triggering of the kill switch, leading to that some certain GMMs can evolve mechanisms to bypass or deactivate the kill switch. Premature self-destruction may result in the microbe not finishing its intended task; late self-destruction may result in the organisms escaping their intended target and contaminating the environment or may even provide the organisms enough time to “evolve” past the kill switch, rendering it ineffective. (Rottinghaus et al., 2022). This potential evolutionary escape poses risks to containment and necessitates the development of more robust and multiple safeguard strategies. Moreover, there is a need to improve the precision and reliability of kill switches. The induction mechanisms of some kill switches may lack specificity, leading to unintended effects or off-target consequences. In this regard, the utilization of advanced genetic engineering tools, such as CRISPR/Cas systems, can enable more precise control and activation of kill switches (Hsu et al., 2014).
Existing limitations abound in the existing methods for GMM leakage containment, and even current kill switch technologies often rely on a single induction factor to activate, which can facilitate the evolution of mechanisms in GMMs to bypass or disable the kill switch. This potential for evolutionary escape poses a risk to the containment efforts. To mitigate the risk of failure associated with the single-inducer kill switch and enhance the robustness of the kill switch mechanisms, we propose to design and construct a "multi-factor inducible self-destructive universal biology chassis." This chassis is aimed to fulfill the experimental and production demands across different areas of genetic engineering and synthetic biology, while optimally achieving effective prevention and control of the GGM environmental leakage.
We plan to integrate the gene encoding the lytic protein E from bacteriophage phiX174 into Escherichia coli. Within the bacterium, a gene circuit will be established with lytic protein E as a lethal agent, which is activated by various factors such as temperature, light exposure, lactose, and arabinose. This innovative genetic circuit serves as a kill switch, resulting in a "multi-factor-induced self-destructive synthetic biology chassis." By incorporating multiple inducing factors, our strategy intends to minimize the possibility of engineered organisms evading destruction, thus reinforcing biosafety measures. Following straightforward modifications, our universal chassis can be efficiently deployed in a multitude of applications, including Large-scale industrial production of pharmaceuticals, food, feed and other raw materials, diagnosis and therapy for diseases, environmental monitoring and remediation, control of agricultural pests, diseases and weeds, etc. To validate the efficacy of this universal chassis, based on it, we will develop a biosensor capable of detecting residual antibiotics in the environment. Specifically, our biosensor exhibits fluorescence in the presence of tetracycline-class antibiotics. This novel biosensor enables flexible, rapid, and efficient detection of antibiotic residues.
Han, L. (2004). Genetically Modified Microorganisms. In: Parekh, S.R. (eds) The GMO Handbook. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59259-801-4_2
Lerner, A.; Benzvi, C.; Vojdani, A. The Potential Harmful Effects of Genetically Engineered Microorganisms (GEMs) on the Intestinal Microbiome and Public Health. Microorganisms 2024, 12, 238. https://doi.org/10.3390/microorganisms12020238
Stemke, D.J. (2004). Genetically Modified Microorganisms. In: Parekh, S.R. (eds) The GMO Handbook. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59259-801-4_4
Moe-Behrens GHG, Davis R and Haynes KA (2013) Preparing synthetic biology for the world. Front. Microbio. 4:5. https://doi.org/10.3389/fmicb.2013.00005
Fudan (2020). Design. iGEM.org. https://2020.igem.org/Team:Fudan/Design/kill_switch#FudanTitleWrapper
Bondy-Denomy, J., Pawluk, A., Maxwell, K. L., & Davidson, A. R. (2013). Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature, 493(7432), 429-432. https://doi.org/10.1038/nature11723
Seed, K. D., & Dennis, P. P. (2020). The toxin–antitoxin (TA) systems of mycobacteria. Microbiology Spectrum, 8(2).
Rottinghaus, A.G., Ferreiro, A., Fishbein, S.R.S. et al. Genetically stable CRISPR-based kill switches for engineered microbes. Nat Commun 13, 672 (2022). https://doi.org/10.1038/s41467-022-28163-5
Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262-1278.