Designing microorganisms with precise target recognition capabilities through gene editing to remove specific pollutants or pathogens requires an interdisciplinary approach that combines synthetic biology, genetic engineering, and environmental science. The following are the key steps and technical framework to achieve this goal： --- ### **1. Micro-chassis selection** Select the microorganism suitable for the target scene as the ”host Organism" (host Organism), for example： -**E. coli**(*E. coli*) or **Bacillus subtilis** (*Bacillus subtilis*): Mature laboratory operations and rich gene editing tools. -**Pseudomonas** (*Pseudomonas*) or **Sphingomonas** (*Sphingomonas*): Strong environmental adaptability and natural ability to degrade pollutants. -**Engineering yeast**or **cyanobacteria**: Suitable for light energy-driven or complex metabolic scenarios. --- ### **2. Design of target recognition mechanism** Through gene editing, microorganisms are given the ability to accurately identify targets. Common methods include： -**Surface treatment engineering**： -Modify the membrane proteins of microorganisms (such as chemotactic receptors or adhesion proteins) to specifically bind pollutants (such as heavy metals, organic compounds) or pathogen surface antigens. -For example: design antibody fragments (scFv) or aptamers (Aptamer) to fuse with membrane receptors to target specific molecules. -**Transcription factor sensor**： -Use natural or engineering transcription factors (such as LuxR, TetR family) to activate downstream gene expression after detecting target molecules (such as antibiotics, aromatic hydrocarbons). -For example: the transcription factor BphR, which targets the pollutant polychlorinated biphenyls (PCB), can regulate the activation of degradation genes. -**CRISPR-Cas system**： - Use proteins such as dCas9 or Cas12a as programmable sensors to identify target DNA sequences (such as specific genes of pathogens) through guide RNA (gRNA). -For example: design CRISPR arrays to detect drug-resistant genes (such as NDM-1) in water bodies to trigger lysis or secretion of antibacterial peptides. --- ### **3. Pollutant/pathogen removal mechanism** After identifying the target, the microorganism needs to perform a removal function, which can be achieved in the following ways： -**Metabolic pathway engineering**： -Introduce or optimize enzyme systems that degrade pollutants, such as： - **Petroleum hydrocarbon degradation**: overexpression of cytochrome P450 or oxygenase (oxygenase). -**Heavy metal adsorption**: Expression of metal binding proteins (such as metallothionein) or secretion of polyphosphates. -**Antibacterial active substance**： -For pathogens, design systems that secrete antibacterial peptides (such as nisin), phage lyases, or produce reactive oxygen species (ROS). -**Group collaboration**： -Coordinate the behavior of microbial populations through quorum Sensing (such as the LuxI/LuxR system) to enhance clearance efficiency. --- ### **4. Gene circuit optimization and regulation** -**Dynamic response**: Design a feedback loop or logic gate (AND/OR gate) to ensure that the clearance mechanism is activated only when the target exists to avoid waste of resources. -For example: a dual-input system (pollution detection + environmental pH/temperature conditions) triggers gene expression. -**Signal amplification**: Use cascade reaction or positive feedback loop to enhance the output signal and improve the clearance efficiency. -**Self-destruct mechanism**: Introduce a "suicide switch" (such as a toxin-antitoxin system) to prevent the spread of engineering bacteria after completing the task. --- ### **5. Enhanced security and robustness** -**Environmental adaptability**: Optimize the salt resistance, temperature resistance or resilience of microorganisms to adapt to complex environments (such as wastewater and soil). - **Horizontal gene transfer blocking**: Delete the binding transfer element or introduce the CRISPR interference system to prevent genes from escaping. -**Nutrient dependence**: Design the conditional expression of essential genes (such as amino acid synthesis genes) to limit the survival of microorganisms in a non-target environment. --- ### **6. Experimental verification and iterative optimization** -**Laboratory testing**: Verify the identification efficiency of microorganisms (such as fluorescence reporting system) and removal ability (such as pollutant concentration detection) in a simulated environment. - **Computational modeling**: Use bioinformatics tools (such as COBRA, GEMs) to predict metabolic flux and optimize gene circuit design. -**Field testing**: Gradually promote the application testing from micro-universe experiments to the real environment to assess the ecological impact. --- ### **7. Application scenarios and challenges** -**Typical application**： -Remove heavy metals (such as mercury and arsenic) or organic pollutants (such as pesticides and plasticizers) from water bodies. -Targeted killing of drug-resistant bacteria (such as Pseudomonas aeruginosa) or intestinal pathogens. -**Challenge**： -**Specificity and cross-reaction**: Avoid misidentifying non-target molecules. -**Biosafety**: Prevent the ecological invasion of engineering bacteria or the horizontal transfer of genes. -**Large-scale production**: Low-cost culture and delivery technology (such as immobilized microbial particles). --- ###**Case reference** -**Synlogic engineering bacteria**: Used to degrade phenylalanine