Designing microorganisms with precise target recognition capabilities through gene editing to remove specific pollutants or pathogens is a cutting-edge research direction in the fields of synthetic biology and genetic engineering. This process involves multiple steps and requires a comprehensive use of molecular biology, gene editing techniques, and an in-depth understanding of microbial metabolic pathways. The specific steps are as follows： 1. **Identify target pollutants or pathogens** - **Pollutant analysis**: Learn more about the chemical structure, nature, and form of presence of the target pollutant in the environment. For example, for organic pollutants, it is necessary to clarify the functional groups in their molecular structure, the length of the carbon chain and other information; for heavy metal pollutants, it is necessary to know their types (such as mercury, cadmium, lead, etc.) and their chemical forms (ionic state, complex state, etc.). -**Characteristics of pathogens**: For pathogens, master their biological characteristics, such as the cell wall structure of bacteria, the nucleic acid type (DNA or RNA) and replication method of viruses, the cell morphology and metabolic characteristics of fungi, etc. 2. **Choose a suitable microbial host** -**Natural characteristics**: Select microorganisms with certain environmental adaptability and metabolic diversity as the basis for transformation. For example, some bacteria have strong environmental tolerance and can survive under different temperatures, pH values, and nutritional conditions; some fungi have a preliminary ability to degrade specific types of organic pollutants. -**Safety**: Consider the safety of microorganisms in the environment to avoid them becoming a new ecological threat. Generally choose microorganisms that are non-pathogenic, easy to control, and will not over-proliferate in the natural environment and cause ecological imbalances. 3. **Design target identification element** -**Receptor protein**: The use of gene editing technology to introduce or modify specific receptor protein genes so that they can specifically identify target pollutants or pathogens. For example, design a protein receptor with a high affinity for a particular organic pollutant, and adjust its amino acid sequence to optimize its binding ability to pollutants through reasonable protein engineering design. -**Nucleic acid aptamers**: For some pathogens, nucleic acid aptamers can be designed. Nucleic acid aptamers are single-stranded oligonucleotides screened by exponentially enriched ligand phylogenetic technology (SELEX), which can specifically bind to specific domains of the target pathogen and interfere with its normal physiological functions. 4. **Construct gene editing vector** -**Vector selection**: According to the characteristics of the target microorganism and gene editing needs, select suitable vectors, such as plasmids, bacteriophages, etc. The vector should have a controllable promoter in order to accurately control the expression level of the target recognition element gene; at the same time, there must be a suitable polyclonal site for inserting the target gene. - * *Gene integration**: Accurately clone the designed target recognition element gene onto the vector, and ensure that it is correctly connected to the regulatory element on the vector to form a recombinant gene editing vector. 5. **Import microbial host** -**Transformation method**: Suitable transformation techniques are used to introduce recombinant vectors into selected microbial host cells. Common transformation methods include chemical transformation methods (such as using calcium chloride to treat cells to make them easy to inoculate exogenous DNA), electrical transformation methods (forming micropores in the cell membrane through high-voltage pulses, allowing carrier DNA to enter) and so on. -**Screening positive clones**: Using screening marker genes carried on the vector (such as antibiotics resistance genes), the positive cloned strains that have been successfully introduced into the recombinant vector are selected by culturing on a medium containing corresponding antibiotics. 6. **Gene editing and optimization** -**CRISPR/Cas system**: CRISPR/Cas gene editing technology is used to accurately modify the genome of microorganisms to ensure that the target recognition element genes can be stably integrated into the host genome and expressed correctly. By designing a suitable guide RNA (gRNA), the Cas protein is guided to target a specific location in the cleaved genome and the target gene is accurately inserted. -**Expression optimization**: Optimize the expression of target recognition element genes, adjust the frequency of codon use, optimize promoter strength, etc., in order to improve their expression level and activity, and enhance the ability of microorganisms to recognize target pollutants or pathogens. 7. **Functional verification and performance evaluation** -**Laboratory testing**: Under laboratory conditions, the modified microorganisms are co-cultured with the target pollutant or pathogen, and their removal ability to the target is verified by detecting changes in the concentration of pollutants, the reduction of the number of pathogens, and the generation of related metabolites. -**Environmental simulation**: Simulate actual environmental conditions, evaluate the removal effect of microorganisms on target pollutants or pathogens under different environmental parameters (such as temperature, humidity, light, etc.), and further optimize their performance. 8. **Safety assessment** -**Ecological risk**: Assess the survivability, transmission potential, and potential impact of transformed microorganisms on other organisms in the natural environment to ensure that they do not cause ecological risks. -**Human health**: Detect the potential hazards of microorganisms and their metabolites to human health, such as whether they are toxic, allergenic, etc., to ensure the safety of their application.