Bt Research 2025, Vol.16, No.6, 269-277 http://microbescipublisher.com/index.php/bt 274 2020). This type of configuration enables it to demonstrate a strong lethal ability against mosquitoes and also better adapt to complex ecological environments. Population genomics explains that the reason why these Bt strains can "show their unique skills" is due to the co-evolution of plasmids and chromosomes, along with the "flexible combination" of horizontal gene transfer and content variation (Figure 2) (Meric et al., 2018). These mechanisms fundamentally drive the functional differences among strains and also enable Bt to find its own survival mode in different niches. Figure 2 Phylogeny of 190 genomes and cry toxicity in the Bacillus cereus species complex (Adopted from Méric et al., 2018) 7 Applications in Bt Metabolic Engineering and Plasmid Tool Development 7.1 Roles of plasmids as expression vectors in bt genetic engineering In Bt's genetic engineering, plasmids are almost standard tools. It does not merely "transport genes", but can also maintain high expression to ensure sufficient production of toxins or other metabolic products. Especially those linear particles from prophages have now been well-tuned. They can maintain high copy operation by themselves without applying screening pressure externally, and the efficiency is not low (Zhao et al., 2024). However, relying solely on one system is not enough. To cope with different scenarios, researchers have also made many combinations, such as using plasmids of different copy number mutants to build shuttle vectors specifically for expressing δ -endotoxin. Moreover, the expression level is indeed directly proportional to the copy number. Of course, there is also a saturation point. Excessive expression can instead affect the stability of the system. In terms of operation, nowadays, not only electrotransformation but also transfer methods under natural receptive states are becoming increasingly popular. These methods have made the plasmid introduction of Bt strains more flexible and broadened the "toolbox" of metabolic engineering (Zegeye and Aspholm, 2022). 7.2 Modular plasmid design strategies in synthetic biology frameworks The term "modularization" is almost a "standard mindset" in the field of synthetic biology. It is no longer a novelty to use modular plasmid systems for metabolic regulation on Bt. Researchers developed promoter libraries and signal peptide libraries, one library within another, in order to fine-tune protein expression levels and make the production process controllable and efficient (Zhao et al., 2024). Moreover, nowadays, it's not just about using these tools in a single strain; many self-transfer plasmids with conjugation functions are also being employed, enabling the module to "jump" into other bacteria. This horizontal transfer between strains and even species has significantly increased the iteration speed of Bt strains. Of course, for the entire system to be reliable, mere
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