Bt Research 2025, Vol.16, No.4, 157-167 http://microbescipublisher.com/index.php/bt 158 review of the current application status of Bt strains and the bottlenecks of traditional transformation, this study will focus on introducing the principles and tools of synthetic biology, discuss how to use these cutting-edge means to engineer Bt strains to improve their insecticidal effectiveness, and look forward to the future development direction of this field. 2 The Basic Principles and Tools of Synthetic Biology 2.1 DNA synthesis and modular design One of the core concepts of synthetic biology is to modularly recombination and design biological components at the molecular level. In recent years, the development of artificial DNA synthesis and genome assembly technologies has enabled scientists to synthesize Bt toxin genes or regulatory elements of any sequence and insert them into Bt strains according to a modular approach. Through de novo DNA synthesis, Bt toxin genes can be customized and modified, such as optimizing codons to increase expression, or fusing functional domains from different sources to create chimeric toxin genes (chimera). Modular design is also reflected in the standardization of vectors and components: researchers can use plasmid vectors with a unified interface to combine promoters, toxin genes, terminators and other modules like "Lego" building blocks to build a Bt expression system that meets needs. A modular plasmid was constructed, which synchronously carried multiple Cry toxin genes and regulatory elements, and achieved synergistic expression of multiple toxins after being transferred to the Bt strain at one time (Park et al., 2000). This method avoids the tedious steps of traditional transformation one by one, and significantly improves the efficiency of strain transformation. 2.2 Gene circuit and regulatory components Synthetic biology draws on the concept of electronic engineering to construct "gene circuits" in microorganisms to program and control the expression of target genes. For Bt strains, cleverly designed regulatory elements can achieve on-demand expression of toxin proteins and improve the spatiotemporal specificity and efficiency of toxin synthesis. Commonly used gene circuit elements include inducible promoters, feedback regulatory circuits, counters, etc. Researchers have constructed a loop with population sensing signals as input, which initiates high expression of toxin genes when the Bt bacterial density reaches a certain threshold, thereby achieving intelligent regulation of "high density-high toxicity" in a field environment (Rocha et al., 2012). For example, using a two-component induction system, the expression of the Bt toxin gene can be linked to specific external signals, so that the strain can express toxins in large quantities when it detects entering the insect body or the plant surface. This gene circuit design can reduce the strain's toxin waste in non-target environments and its impact on non-target organisms. 2.3 Application of CRISPR-Cas and other gene editing platforms in Bt transformation CRISPR-Cas gene editing technology has made breakthroughs in microbial engineering in recent years, and has also provided an efficient means for the targeted transformation of Bt strains. The genetic modification of traditional Bacillus is relatively difficult, while the introduction of the CRISPR-Cas9 system makes Bt genome editing more precise and efficient (Soonsanga et al., 2020). By designing specific sgRNAs and binding to Cas9 nucleases, researchers can cause double-strand breaks at pre-local sites in the Bt genome and accurately insert, delete or replace target genes through homologous recombination. This can achieve in-depth modifications that were difficult to complete in the past, such as knocking out non-essential plasmids or virulence-related negative regulatory genes in strains to eliminate metabolic burdens and improve toxin synthesis. In recent years, it has been reported that CRISPR-Cas9 has been used to knock out genes that produce excessive β-exotoxin (toxic to mammals) in Bt strains at one time, thereby building a safer engineering strain (Higgins et al., 2024). The CRISPR tool is also used for genomic insertion of Bt strains: for example, integrating exogenous toxin genes onto Bt chromosomes to obtain genetically stable, highly yielding strains. In addition to Cas9, new nucleases such as Cas12a have also been introduced into Bt, expanding the range of editable sequences. 3 Molecular Mechanism and Target Modification of Bt Toxin 3.1 Structure and mechanism of Cry, Cyt and Vip toxins The insecticidal activity of Bt mainly comes from the δ-endotoxins it produces during the formation of bud cells,
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