The idea of SRBioQuencher is originally deviated from a news about a child who died during Chinese New Year after playing with firecrackers near a manhole cover and causing an explosion. This news brought our attention to the harmful gases in city sewers that we would normally ignore. After research, we found that hydrogen sulfide, as the main harmful gas in sewers, is toxic and has an irritating odor in addition to causing fire accidents. It not only threatens the health of sewer workers, but also tends to cause corrosion of sewer pipes.
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Hazards of Hydrogen Sulfide
The traditional treatment is using a large amount of bactericides to reduce the number of sulfate-reducing bacteria (SRB), the main producers of hydrogen sulfide in sewers, thereby reducing the release of hydrogen sulfide. The use of highly toxic bactericides has raised concerns about their potential health, safety, and environmental impacts.
At the same time, the biofilm provides a substrate for SRBs to adhere to, facilitating their attachment to surfaces such as sewers and the formation of stable bacterial communities. This is beneficial for the long-term colonization of SRBs in sewer pipes.
Currently, there is no good solution to the problem of biologically harmful gases in urban sewers, and this is what SCU-China 2023 hopes to do to build a better world.
Let's restate the current problem we're experiencing:
To handle these problems, we apply cocktail therapy to SRBioQuencher by endowing it with the ability to eliminate the SRB biofilm, inhibit the formation of the SRB biofilm, and kill SRB.
Meanwhile, Apart from the functional modules mentioned above, we modified the chassis due to the special characteristics of urban sewer environment. We designed a predator model-based suicide system for biosafety, and introduced thioredoxin oxidase (SQR) into the chassis to avoid the release of H2S produced by the chassis bacteria themselves.
Fig.1 Project Overview
The matrix of SRB biofilms is primarily composed of exopolysaccharides (EPS). To address existing biofilms, we tested the efficacy of two glycosidases, DspB and DisH, in disrupting the structure of SRB biofilms, in order to select a more suitable strategy for destroying SRB biofilms. The sites of action of these two enzymes are the glycosidic bonds of EPS surface polysaccharides. We hope to weaken the compact structure of the biofilms that these enzymes can help destroy, making the biofilms fragile and easily disrupted by flowing water, while simultaneously destroying the anaerobic environment in which the SRBs thrive, thereby reducing the number of SRBs. We also supplement this approach with the capability of engineered bacteria to express surfactin, a biosurfactant, to further inhibit SRB growth.
We also hope to prevent the formation of SRB biofilms from within the biofilm through quorum quenching. The genes related to biofilm formation are controlled by the bacterial quorum sensing system (QS). As a cell-density-dependent microbial communication system, its signaling molecules are autoinducers (AHLs). Therefore, by degrading AHL molecules, we can disrupt the QS sensing system of SRBs, thereby interfering with the activation process of SRB biofilm formation. To achieve this goal, we introduced the N-acyl homoserine lactonase AiiA and MomL into engineered E. coli, enabling the degradation of a variety of AHL signaling molecules.
Treatments Repressing the Biofilm
Although SRB could be removed by the scouring of water flow after disrupting the biofilm of SRB, in the modeling, we concluded that additional repressing module was needed to enable SRB to achieve an oscillation decrease with the engineered bacteria. Therefore, we first expressed a broad-spectrum AMP to inhibit the growth of SRB. On that basis, a degradable connector based on disulfide bond was then designed to connect the antimicrobial peptide with the two enzymes in the form of fusion proteins to realize the safe secretion of the antimicrobial peptide and to produce certain targeting effects on the SRB microenvironment, so as to avoid the AMP from negatively affecting the engineered bacteria themselves.
AMP fusion protein release strategy
Since the engineered bacteria in this project will ultimately be released into the environment, we have constructed a suicide system based on a predator model to prevent genetic contamination.
The engineered bacteria can perceive the concentration of SRB autoinducers, thereby regulating the activation and deactivation of the downstream suicide mechanism. This establishes a predator-prey relationship between the engineered bacteria and SRBs, ensuring that the engineered bacteria can function effectively over the long term. At the same time, this mechanism also enables the engineered bacteria to automatically initiate a suicide program after leaving the sewer system and entering the main waterway, ensuring the biosafety of the engineered bacteria and reducing the possibility of horizontal gene transfer.
Predator-prey model based suicide system
It has been shown (Liu ,et.al, 2019) that E. coli also secretes hydrogen sulfide to be used as an autoinducer, in order to avoid the secretion of H2S produced by engineered bacteria to the environment, we introduced thioredoxin oxidase (SQR) in engineered bacteria to immobilize free hydrogen sulfide into the indiffusible hydrogen polysulfide
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Scarascia G, Wang T, Hong PY. Quorum Sensing and the Use of Quorum Quenchers as Natural Biocides to Inhibit Sulfate-Reducing Bacteria. Antibiotics (Basel). 2016 Dec 15;5(4):39. doi: 10.3390/antibiotics5040039
Tang K, Su Y, Brackman G, Cui F, Zhang Y, Shi X, Coenye T, Zhang XH. MomL, a novel marine-derived N-acyl homoserine lactonase from Muricauda olearia. Appl Environ Microbiol. 2015 Jan;81(2):774-82. doi: 10.1128/AEM.02805-14. Epub 2014 Nov 14.
Zhu L, Poosarla VG, Song S, Wood TL, Miller DS, Yin B, Wood TK. Glycoside hydrolase DisH from Desulfovibrio vulgaris degrades the N-acetylgalactosamine component of diverse biofilms. Environ Microbiol. 2018 Jun;20(6):2026-2037.
