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Applications | WHU-China - iGEM 2023
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  • Part 1: IBD Diagnosis
  • Part 2: PA infection Diagnosis
  • Part 3: Macrolides detection in aqueous environment
  • Part 4: PCA monitoring
  • References
    • To top

    The CRISPReporter, due to its remarkable recording capacity and potential to sense diverse signals, holds promise for a multitude of applications. We intend to explore the use of other promoters by replacing the EL222 promoter in various domains: Pphsa to diagnose IBD, lasR & P luxR system to diagnose Pseudomonas aeruginosa infection, MPhR-pMphR to detect macrolides and Pcau to monitor PCA. On this page, we describe our concept design for these applications. We hope that we can inspire others to come up with other ideas about applying CRISPReporter to the real world.

    Fig 1.Proposed applications of CRISPReporter driven by difference promoters

    Part 1: IBD Diagnosis

    Inflammatory Bowel Disease (IBD) encompasses a group of chronic inflammatory disorders that affect the gastrointestinal tract, leading to symptoms such as diarrhea, hematochezia, abdominal discomfort, and fatigue.[1] These symptoms can significantly impair the physical and psychosocial well-being of patients.

    IBD has a high prevalence worldwide and has displayed a growing trend in developing countries. For instance, the morbidity of IBD is about 1.74 per 100,000 person-years in China.[1] IBD is characterized by its chronic nature and recurrent episodes, causing intermittent distress and substantial financial burden to patients over many years.

    IBD comprises ulcerative colitis (UC), Crohn's disease (CD), and indeterminate colitis (IC, i.e., IBD that cannot be definitively diagnosed as UC or CD).[2,3] After consulting with an IBD professor, we chose to focus on the diagnosis of UC due to the feasibility of conducting animal experiments. (Disclaiming: We did not conduct any animal experiments during the competition.) Dextran Sulfate Sodium (DSS) can induce direct damage to the colonic mucosa and trigger an inflammatory response in animal models of UC, whereas the construction of CD animal models involves more complex methods.[2]

    Presently, diagnostic methods for IBD include colonoscopy and endoscopy.[4] However, these traditional procedures are invasive and can cause significant discomfort to patients. Moreover, they are incapable of recording multiple episodes of IBD, meaning that physicians must rely on patient narratives to determine disease severity—an approach that lacks precision.

    Our aim is to develop an innovative non-invasive diagnostic method for IBD based on engineered bacteria capable of recording multiple episodes in patients with IBD. Once colonized in the intestinal tract, these bacteria can detect the concentration of thiosulfate—a biomarker for IBD pathogenesis—in the intestine over an extended period.[5]

    We designed to replace the blue-light-inducible promoter EL222 in the recording system with the ThsR/ThsS two-component system from Shewanella halifaxensis to respond to thiosulfate (Fig 2). ThsS is a histidine kinase that is expressed constitutively and integrated into the cell membrane of engineered E. coli. In the presence of thiosulfate, ThsS phosphorylates the constitutively expressed cytoplasmic response regulator ThsR. The phosphorylated ThsR then activates our recording system. [6]

    Fig 2. Mechanism of ThsR/ThsS two-component system (Adapted from Daeffler KN et al., Mol Syst Biol, 2017)

    Patients would orally consume the engineered bacteria that are encapsulated with an enteric coating. After reaching the gastrointestinal tract, the coating gradually degrades, releasing the engineered bacteria. These bacteria then colonize the intestines, and as the concentration of intestinal thiosulfate increases, the recording system is activated. The frequency of IBD attacks during this period can be determined by detecting signals from the engineered bacteria excreted in feces.

    Part 2: PA Infection Diagnosis

    Pseudomonas aeruginosa (PA) is a major opportunistic pathogen, considered one of the most common hospital-acquired pathogens worldwide.[7] The human intestinal tract serves as a common harbor of PA, which has the potential to induce infections in other individuals via feces, contaminated medical devices, and the environment.[8] They may be disseminated to other body sites and cause endogenous infection, especially in immunocompromised patients.[9]

    PA exhibits both inherent and acquired resistance mechanisms, making it resistant to most antibiotics.[10] Once colonization occurs, it becomes challenging to eradicate and may lead to recurrent episodes of infection. Currently, applied detection methods for PA like PCR, and isothermal amplification techniques are incapable of detecting relapse time.[11] Our product can fill this gap in the field by simply changing the promoters.

    We chose 3OC12HSL, a quorum-sensing molecule at the early stage of PA infection, as our biomarker. The lasR transcription factor binds to the PluxR promoter in the presence of 3OC12HSL, thereby activating transcription (Fig 3).[12]

    Fig 3. Mechanism of lasR & PluxR system (Adapted from Saeidi, N., et al., Molecular Systems Biology, 2011)

    We designed to replece the EL222 promoter with this system, enabling the recorder to detect 3OC12HSL.Similar to the method described in part 1, our engineered bacteria will be encapsulated in an enteric coating for delivery into the intestine[13] to facilitate real-time monitoring. When the concentration of 3OC12HSL in the intestine surpasses the threshold, recording is triggered, indicating a potential attack. The frequency of PA attacks during this period can be determined by detecting signals from the engineered bacteria excreted in feces.

    Part 3: Macrolides Detection in Aqueous Environment

    Macrolide antibiotics have recently seen extensive use in clinical prophylaxis and treatment.[14] However, these antibiotics have gradually permeated into surrounding water bodies and other media, posing potential risks to both the environment and human health. [15, 16] The harsh environment can further stimulate certain strains to mutate into drug-resistant bacteria, disrupting the ecological balance. Therefore, the detection of macrolide antibiotics in aquatic environments is of paramount importance.

    Several conventional methods exist for detecting antibiotic pollutants in water environments, including chromatography, mass spectrometry, capillary electrophoresis, optical detection, and electrochemistry.[17] While these methods have their merits, they also present limitations such as being labor-intensive, time-consuming, and necessitating prior bacterial identification.[17]

    With the continuous advancement in science and technology, new detection methods are emerging. Biosensor-based detection, for instance, holds promising potential. It can achieve high sensitivity, rapid speed, sustainable detection, and low cost by utilizing biological molecules or cells that can recognize and respond to antibiotic pollutants.[17,18,19]

    We intend to apply CRISPReporter to environmental monitoring. For the design of the system to respond to macrolides, we referred to 2020 Aalto-Helsinki----SINISENS.

    Fig 4. Mechanism of MPhR-pMphR

    The design comprises three components: the macrolide-related promoter, pMphR; the repressor of pMphR, MPhR; and the macrolide resistance gene, ermC[20] (Fig4, adapted from Aalto-Helsinki, iGEM, 2020). In the absence of macrolides, MPhR binds to pMphR, inhibiting the expression of downstream genes. When macrolides are present in the sample, two macrolide molecules bind to MphR. This binding releases MPhR from pMphR, allowing the transcription of downstream genes.

    We designed to replace the EL222 promoter in our original design with pMphR so that our biosensor can sense the concentration of macrolides and record the information in DNA over an extended time.

    Our environmental detection chassis will be encased in a specialized material and deployed into the aquatic environment for real-time monitoring (Fig5). Once the concentration of macrolides in the water surpasses the minimum response threshold of pMphR, recording will be triggered, storing long-term information of macrolides pollution in the water. By retrieving the engineered bacteria, we can report on macrolide contamination through quantitative PCR. Our system introduces a novel possibility for detecting low concentrations of antibiotics in water.

    Fig 5. Product concept

    Part 4: PCA monitoring

    Plastics with desirable properties have been industrially produced over the past century and widely incorporated into consumer products.[21] Many of these products exhibit remarkable persistence in the environment due to the lack or low activity of catabolic enzymes capable of degrading their plastic constituents.

    In particular, polyesters with a high proportion of aromatic components, such as polyethylene terephthalate (PET), are chemically inert, resulting in resistance to microbial degradation.[22,23]

    Fig 6. Condition of PET contamination
    The production of polymers in 2018 is reported to be approximately 359 million tonnes, and it is predicted that in the next 30 years, the production of these materials will triple.[24,25] It is estimated that PET accounts for 18% of the global production.[26,27] A minor fraction of this PET waste is recycled, while the rest is left in the environment regardless of its destructive effects.[27]
    Fig 7. Limitations of current measurement methods
    Biodegradation has emerged as a focal point of research. However, the measurement of degradation is short of attention. Currently used measurement methods each have their limitations. Gravimetry operates discontinuously because the PET sample has to be removed from the enzyme solution and dried to be measured, leaving the analysis with a low temporal resolution. It is also labor-intensive because most steps are manually performed.[28] Optical methods such as spectrophotometry, fluorescence spectroscopy, and turbidimetry require costly equipment.

    The proposed product aims to achieve cost-effective continuous measurement of PET degradation by changing the promoters. We chose PCA, a downstream byproduct of TPA metabolism, as our biomarker.[29] In the absence of PCA, the transcription factor PcaU binds to the operator region, inhibiting its own transcription and a target transcript. When PCA is present, PcaU releases the region, and transcription of the target transcript starts (Fig8).[30]

    Fig 8. Mechanism of PcaU (Ramesh K.J et al, 2014)

    We designed to replace the EL222 promoter with this system, enabling the recorder to detect PCA. Similar to the method described in part 3, engineered bacteria will be encapsulated in a specialized material and deployed into the aqueous environment where PET is recycled to facilitate real-time monitoring. When the concentration of PCA in the water surpasses the degradation level, our monitor will keep a record of it, suggesting that the recycling process is done. By retrieving the engineered bacteria, we can report on the degree of degradation through qPCR. Our system introduces a novel approach for continuously measuring PET degradation at low costs.

    References

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