As described in the project description, our goal is to construct a genetically engineered bacterium that possesses both defensive and offensive capabilities, allowing it to stably occupy a niche in specific environments and prevent the invasion and survival of other pathogenic bacteria. However, the application of engineered bacteria poses two major biosafety concerns: first, the potential for engineered bacteria to accidentally leak into the environment, leading to ecological imbalance; and second, the potential risk of horizontal gene transfer, which could result in gene dissemination to wild strains, impacting natural biodiversity.
This year's iGEM competition represents the first phase of this project, primarily focusing on monitoring the growth curves, motility assessments, biofilm formation, capsular polysaccharide quantification, inhibition zone observation, and inter-strain competition experiments of the involved and related modified strains. For safety considerations, we have selected Acinetobacter baylyi ADP1, which possesses an effective Type VI secretion system (T6SS) and remote attack capabilities, to replace pathogenic bacteria in testing the defensive and offensive abilities of genetically modified Escherichia coli Nissle 1917 strains. Additionally, we used commonly employed laboratory strains of E. coli, namely BL21, BL21(DE3), and DH5a as experimental strains.
To this end, we designed three systems in the chassis strain: a defense system, an offense system, and a suicide system. The resulting engineered bacterium exhibits strong defensive and offensive capabilities and is programmed to undergo self-destruction after a certain period, thereby reducing biosafety risks.
In the defense system, we aimed to enhance the bacterium's structural components—fimbriae, capsular polysaccharide, and cell membrane—from the outermost layer to the innermost to better withstand attacks from bacterial pathogens.
As shown in Figure 1, recent studies in Cronobacter malonaticus have demonstrated that the ΔfimE mutant can overexpress Type I fimbriae, forming microcolonies that protect cells from T6SS-mediated contact-dependent killing, thereby enhancing survival. In our project, we knocked out the fimE gene in E. coli Nissle 1917 to increase fimbrial length, improving resistance to pathogenic attacks.
Notably, FimH, which encodes a mannose-specific adhesin located at the tip of the fimbriae, mediates adhesion to mannose-glycosylated receptors on cell surfaces. Inter-bacterial killing assays suggest that the T6SS resistance exhibited by the fimE mutant is more likely attributed to microcolony formation via FimH rather than being driven by fimbriae-mediated single-cell spatial separation. Additionally, reports indicate that FimH plays a crucial role in biofilm formation. Therefore, we constructed a plasmid to overexpress fimE in E. coli Nissle 1917 to enhance bacterial adhesion and biofilm production, thereby supporting the function of the defensive engineered bacteria.
Introducing spatial separation between the target cells and attacking cells is an effective mechanism for resisting T6SS contact-dependent attacks. This spatial separation can be triggered by the production of extracellular polysaccharides (EPS), creating a physical barrier or biofilm that leads to the formation of microcolonies, protecting the cells located at the center. Furthermore, at the interface between the attackers and target cells, dead cells and debris can also form a barrier to enhance protective effects. In recent years, several iGEM projects have focused on capsular polysaccharides, such as those from the teams at Xi'an Jiaotong University in 2020 and 2022, and Fudan University in 2023. In these projects, increasing the production of extracellular polysaccharides was primarily achieved through overexpression of pgmA and galU genes, enabling the strains to produce more EPS. In our project, we aim to have more capsular polysaccharides adhere to the bacterial surface for better protection. Recent research has shown that the flagellar protein FliC in E. coli Nissle 1917 (EcN) can bind to heparin. Therefore, we overexpressed FliC in E. coli Nissle 1917 using plasmids and supplemented with heparin or overexpressed pgmA and galU genes to produce excess capsular polysaccharides, thereby increasing the thickness of the capsular polysaccharides and enhancing the defensive capabilities of the engineered bacteria.
In addition to the common methods previously used to increase capsular polysaccharide production, this project also approaches the problem from the perspective of capsular polysaccharide transport. By attempting to increase the extracellular polysaccharide content, we aim to enhance the strain's defenses while effectively reducing the metabolic costs of producing more polysaccharides. Improving the strain's reproduction rate or other offensive and defensive abilities would be a significant enhancement.
Based on previous studies (Figure 3), we selected the KpsT and KpsE genes related to capsular polysaccharide transport for plasmid introduction into E. coli Nissle 1917 (EcN). This allows for comparative studies of extracellular polysaccharide content and bacterial defense capabilities, aiming to screen for more engineered bacterial strains with high defensive potential and further optimize strain performance.
Escherichia coli 1917 or Escherichia coli Nissle 1917 (DE3) possesses stronger offensive capabilities and environmental adaptability compared to other established engineered strains such as E. coli BL21, BL21(DE3), and DH5α. Importantly, Escherichia coli Nissle 1917 has long-range attack abilities, as mentioned in the “description” section, which include the capacity to inhibit various pathogenic bacteria. Despite the synthetic biology toolbox accumulating numerous functional modules that enable the VI type secretion system to be applied in E. coli BL21(DE3) and DH5α, the relatively complete T6SS system in Escherichia coli Nissle 1917 holds greater potential for optimizing offensive capabilities, allowing it to target a wider range of pathogenic bacteria and fungi. Additionally, Escherichia coli Nissle 1917 has two independent CDI systems awaiting modification.
Microcins are a class of low-molecular-weight, ribosomally synthesized, and highly stable bacteriocins involved in bacterial competition. Compared to contact-dependent inhibition systems, the modification of microcins in E. coli is relatively straightforward. We overexpressed the antimicrobial peptide-related mcmA gene, along with its immunity protein gene, in Escherichia coli Nissle 1917 (DE3) using plasmids to perform inhibition zone assays and inter-bacterial competition experiments to verify the enhanced aggressiveness of Escherichia coli Nissle 1917 (DE3). Simultaneously, we introduced the mchB gene and its immunity protein gene mchI from Escherichia coli Nissle 1917 into E. coli BL21(DE3) to assess the attack capabilities of the peptide antimicrobials related to the mchB gene in BL21(DE3).
Microbial genomes encode a rich arsenal of interbacterial weapons. The widely distributed type VI secretion system (T6SS) has been shown to play a crucial role in competitive colonization within host ecological niches. Through genomic analysis, we discovered that Escherichia coli Nissle 1917 (EcN) possesses a complete T6SS system, although no studies have yet reported its specific offensive capabilities. To investigate this, we overexpressed the secretion protein gene hcp related to the T6SS system using plasmids and employed fluorescence tagging to observe whether fluorescent proteins were secreted extracellularly after induction. Notably, it has been established that the genome of the T6SS-positive Escherichia coli 042 contains the tagh gene, which is absent in EcN and plays a critical regulatory role in the T6SS of Vibrio cholerae. Therefore, we introduced the tagh gene from E. coli 042 into EcN using plasmids to examine its impact on EcN's aggressiveness. According to preliminary literature and big data analysis, a highly lethal CRAB (Carbapenem-resistant Acinetobacter baumannii) strain exists in Guangdong province. We established a database related to Acinetobacter baumannii that enables rapid identification of missing immunity genes in specific regions, thereby facilitating the identification of weapons to combat it and laying a foundation for further weapon enhancement in Escherichia coli Nissle 1917.
Contact-dependent growth inhibition (CDI) is a competitive mechanism identified in various strains, including Burkholderia, Dickeya, E. coli, and Yersinia. The classic CDI system consists of three genes: cdiB, cdiA, and cdiI. The CdiB protein encoded by the cdiB gene is a conserved β-barrel protein responsible for the secretion of CdiA; the cdiA gene encodes the CdiA protein, which includes a conserved N-terminal domain and a variable C-terminal toxic domain (CdiA-CT). The immunity protein CdiI binds to and inactivates the toxic protein CdiA-CT. Two relatively independent CDI systems have been identified in EcN, but mutations in the CdiB gene may affect CdiA's functionality. Our initial design aimed to replace the mutated CdiB gene in EcN to introduce a functional CdiB, but due to time constraints, this project first introduced a normally functioning CdiB into EcN via plasmids to observe whether EcN's aggressiveness would be enhanced.
Considering human and environmental safety, we aim to design a suppression switch for our engineered strain. We referenced the temperature-controlled suppression switch design from the 2023 SHSID-China team and made modifications. The bacterial toxin-antitoxin system CcdB-CcdA provides a mechanism for controlling cell death and dormancy. The antitoxin protein CcdA is a homodimer composed of two monomers, containing a folded N-terminal region and an intrinsically disordered C-terminal arm. The C-terminal arm of CcdA binds to the toxin CcdB, preventing CcdB from inhibiting DNA gyrase, thereby avoiding cell death. When the environmental temperature of the engineered strain is below 37°C, the control switch remains in its normal state, allowing for the expression of CcdA at a slightly lower relative level than CcdB. When the engineered strain comes into contact with the human body and the environmental temperature rises to 37°C, the control switch shuts down, resulting in the non-expression of the CcdA gene and the relative dominance of CcdB, disrupting the balance between the toxin and antitoxin, leading to the death of the engineered strain. To ensure that the accumulation of CcdB can effectively exert its lethal effect within the time frame, we will adjust its expression frequency by replacing the synonymous codons in the CcdB sequence, thus controlling the accumulation of toxins in the engineered strain within the ideal time frame. Considering the variability in environmental temperatures, we conducted a temperature survey of hospital environments in Shenzhen to ensure that the temperature-controlled suppression switch would not affect the feasibility of the engineered strain due to seasonal temperature variations.
In most parts of China, toilets are widely utilized with a toilet tank-based flushing system. For this system, our team has innovatively developed a hybrid freeze-dried strain of Gel Beads and improved the design of the tank structure of traditional flushing toilets. Whenever the user presses the flush button, the beads will be released from the special tank and form aerosols in the air with the water flow, which will occupy the ecological niche and continuously inhibit the pathogenic bacteria. In addition, the system naturally generates gravitational potential energy during the flushing process, which is cleverly captured and utilized in the system. Through careful design, the water flow is able to push the piston inside the pipe, which in turn exerts pressure on the paddle motor and drives it to rotate. As the motor rotates, the mechanical energy is efficiently converted into electrical energy, which is ultimately used to provide constant and stable power to the product storage tanks, ensuring that the tanks are maintained at the desired temperature levels.
[1] Margot Marie Dessartine, Artemis Kosta, Thierry Doan, Éric Cascales, Jean-Philippe Côté. Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks. mBio. 2024 Apr 10;15(4):e0255323.
[2] Neamati Foroogh, Moniri Rezvan, Khorshidi Ahmad, Saffari Mahmood. Structural and functional characterization of the FimH adhesin of uropathogenic Escherichia coli and its novel applications. Microb Pathog. 2021 Dec;161(Pt B):105288.
[3] Laurel K Mydock-McGrane, Thomas J Hannan, James W Janetka. Rational design strategies for FimH antagonists: new drugs on the horizon for urinary tract infection and Crohn's disease. Expert Opin Drug Discov. 2017 Jul;12(7):711-731.
[4] Steven J Hersch, Kevin Manera, Tao G Dong. Defending against the Type Six Secretion System: beyond Immunity Genes. Cell Rep. 2020 Oct 13;33(2):108259.
[5] Elisa T Granato, William P J Smith, Kevin R Foster. Collective protection against the type VI secretion system in bacteria. ISME J. 2023 Jul;17(7):1052-1062.
[6] Bram Lories, Stefanie Roberfroid, Lise Dieltjens, David De Coster, Kevin R Foster, Hans P Steenackers. Biofilm Bacteria Use Stress Responses to Detect and Respond to Competitors. Curr Biol. 2020 Apr 6;30(7):1231-1244.e4.
[7] Guixia Yang, Lingkang Yang, Xianxuan Zhou. Inhibition of bacterial swimming by heparin binding of flagellin FliC from Escherichia coli strain Nissle 1917. Arch Microbiol. 2023 Jul 15;205(8):286.
[8] Shan Hu, Siyan Zhou, Yang Wang, Wuxia Chen, Guobin Yin, Jian Chen, Guocheng Du, Zhen Kang. Coordinated optimization of the polymerization and transportation processes to enhance the yield of exopolysaccharide heparosan. Carbohydr Polym. 2024 Jun 1:333:121983.
[9] Yang Cui, Tong-Tong Pei, Xiaoye Liang, Hao Li, Hao-Yu Zheng, Tao Dong. Heterologous Assembly of the Type VI Secretion System Empowers Laboratory Escherichia coli with Antimicrobial and Cell Penetration Capabilities. Appl Environ Microbiol. 2022 Oct 11;88(19):e0130522.
[10] Fernando Baquero, Val F Lanza, Maria-Rosario Baquero, Rosa Del Campo, Daniel A Bravo-Vázquez. Microcins in Enterobacteriaceae: Peptide Antimicrobials in the Eco-Active Intestinal Chemosphere. Front Microbiol. 2019 Oct 9:10:2261.
[11] Yuexia Luna Lin, Stephanie N Smith, Eva Kanso, Alecia N Septer, Chris H Rycroft. A subcellular biochemical model for T6SS dynamics reveals winning competitive strategies. PNAS Nexus. 2023 Jun 13;2(7):pgad195.
