1. CotAgold: The basic structure of this part is derived from the laccase of Bacillus subtilis, which can degrade aflatoxins. Relevant literature has introduced three site-directed mutations that significantly improve its degradation efficiency and stability while reducing its environmental requirements. We further optimized it for expression in our chassis organism, Lactobacillus rhamnosus ATCC 7469.

2. PGlu+MazF: This is the suicide gene we designed, which utilizes GluKiller and MazF to trigger the expression of intracellular toxins when the external glucose concentration decreases, preventing the engineered strain from escaping. We further optimized it to ensure that after cell death, the cells do not undergo lysis, preventing the release of intracellular toxins and ensuring biosafety.

3. G8-Gluc: We linked the G8 nanobody and Gluc together using a protein linker to assemble a fusion protein. G8 specifically binds to AFB1, and upon binding to AFB1, Gluc oxidizes the substrate coelenterazine, producing blue fluorescence. [1]

4. paT7P-1: This part is derived from a T7 RNA polymerase and two VVD domains. Based on literature references, we split the T7 RNA polymerase at position 564-565 and linked each fragment to a VVD domain[2], forming two fusion proteins that coexist in the cell. Upon receiving blue light, VVD forms homodimers, allowing the two parts of the T7 RNA polymerase to bind together and restore its biological activity. We further optimized it for expression in our chassis organism, Lactobacillus rhamnosus ATCC 7469.

5. Fine control device: We designed a Fine control device using paT7P-1, G8-Gluc, and the T7 promoter. A T7 promoter was added upstream of the target gene to be expressed, preventing the bacterial RNA polymerase from initiating its expression in normal conditions. G8-Gluc is secreted into the extracellular environment, where it specifically detects and binds to AFB1. Afterward, Gluc oxidizes coelenterazine, emitting blue fluorescence[1]. The blue fluorescence acts as a signaling molecule that enters the cell. Upon receiving the signal, the VVD domain undergoes a conformational change, causing the protein expressed by paT7P-1 to form homodimers, restoring the activity of the split T7 RNA polymerase. The reactivated T7 RNA polymerase then recognizes and binds to the T7 promoter, initiating the expression of the target gene.

This device achieves precise and autonomous regulation of the expression of specific substances. We also provide a variety of nanobodies that can recognize different substances.

For optimal regulatory performance, it is best to transfer all vector plasmids into the same bacterial strain.

We have developed a large number of components that can be used by future teams, and assembled a sophisticated regulatory device using these components that can specifically identify certain substances in the environment and regulate gene expression.

We also provide a variety of different nanobodies that can recognize different substances to initiate regulation.