Designing microorganisms with precise target recognition capabilities through gene editing technology to remove specific pollutants or pathogens is a complex process involving synthetic biology, genetic engineering, and environmental science. The following are the systematic steps and key technical points to achieve this goal： --- ### **1. Design of target recognition system** ####**1.1 Biosensor development** -**Receptor protein/RNA aptamer**： Programmable DNA binding proteins such as CRISPR-dCas9 (Cas9 without shear activity) or zinc finger protein (ZFP) are used to bind the sensing domains of specific pollutants (such as heavy metal binding proteins and aromatic hydrocarbon receptors). *Example*: Design a MerR protein promoter that targets mercury ions (Hg20) to drive the expression of downstream degraded genes. -**RNA switch** (Riboswitch) or aptamers： Regulation of gene expression (such as aptamers for antibiotics or organic pollutants) by inducing conformational changes through small molecule binding. ####**1.2Signal conduction and amplification** -**Two-component system** (such as EnvZ/OmpR for bacteria)： The pollutant recognition signal is converted into a transcriptional regulatory signal, which drives strong promoter (such as T7, Ptac) to activate downstream pathways. --- ### **2. Pollutant degradation/neutralization module** ####**2.1 Enzyme pathway design** -**Heterologous expression of degrading enzymes**： -For organic pollutants: introduce oxygenases (such as P450), dehalogenases (degrading PCBs) or hydrolases (decomposing plastics). -For heavy metals: express metal chelating proteins (such as metallothionein) or reductases (reduce Cr3为 to low toxicity Cr3Cr). -**Optimization of metabolic pathways**： Increase the supply of cofactors (NADH/NADPH) and enhance enzyme activity through metabolic engineering (such as MAGE orCRISPRi regulation). ####**2.2 Targeted removal of pathogens** -**Antibacterial peptide or phage tail silk protein**： Design antibacterial peptides that target specific pathogen surface markers (such as LPS or flagellin), or modify phage tail silk proteins to achieve specific binding. --- ### **3. Gene editing tool selection** - **CRISPR-Cas9/Cas12a**： It is used to accurately knock in degraded genes or regulatory elements; Base Editor/Prime Editor can avoid DNA double-strand breaks. -**Synthetic gene circuit**： Construct AND/OR logic gates (such as activation only at high pollutant concentration + low pH) to improve specificity. --- ### **4. Safety and controllability design** ####**4.1Biological Containment strategy** -**Nutrient deficient strains**： Delete essential genes (such as dapA) and rely on exogenous supplementation to limit spread. -**Suicide switch**： Introduce temperature-sensitive plasmids or toxin-antitoxin systems (such as ccdB/ccdA) to self-destruct after completing the task. ####**4.2 Prevention of horizontal gene transfer** -Delete the binding transfer element (such as oriT) and use a genomic integration (non-plasmid) expression system. --- ### **5. Experimental verification and optimization** -**In vitro test**： Verify the sensor sensitivity and degradation efficiency in a microfluidic chip or analog environment. -**Model prediction**： Use COBRA or FBA (flux balance analysis) to optimize the distribution of metabolic flux. -**Adaptive evolution**： Screen high-efficiency strains under stressful conditions (such as high pollutant concentrations). --- ### **6. Application scenarios and challenges** -**Environmental restoration**： Target oil spills (Pseudomonas that express alkane hydroxylase) or agricultural run-off (engineering bacteria that degrade nitrates). -**Medical application**： Intestinal engineering bacteria (such as Escherichia coli Nissle 1917) target the removal of drug-resistant pathogens (such as Clostridium tough). -**Challenge**： Ecological risk assessment, long-term stability, competition with indigenous microorganisms. --- ###**Case reference** -**Treatment of arsenic pollution**： The large intestine was modified to express arsenic oxidase (aioA) and arsenic binding protein (ArsR) to realize the detection and transformation of As3. -**Pathogen detection**： Engineering bacteria based on Quorum Sensing (such as Vibrio fischeri) emit light when encountering pathogens. --- Through interdisciplinary and iterative optimization, gene-edited microorganisms can become a powerful tool for precise environmental restoration and medical intervention, but biosafety ethics must be strictly followed.