In our project, we utilized RED-mediated homologous recombination to knock out the guaB gene and introduce a resistance gene. Specifically, through RED homologous recombination, we first designed a linear DNA fragment containing the upstream and downstream regions of the guaB gene and inserted a resistance gene (KanR) in between. Then, this fragment was introduced into E. coli BW25113 carrying the pkd46 plasmid via electroporation. By inducing the expression of RED recombinase from the pkd46 plasmid at 37°C, the linear DNA fragment underwent homologous recombination with the target genomic fragment, resulting in the replacement of the guaB gene. Finally, colony PCR confirmed the successful knockout of the guaB gene and its replacement with the resistance gene.

RED-mediated homologous recombination is a gene knockout method based on the λ phage RED system. The primary steps include designing linear DNA fragments with 50-100 base pair homology arms, which typically contain a resistance gene for selection. These fragments are introduced into the host strain expressing RED recombinase (commonly E. coli strains carrying the pkd46 plasmid) through electroporation. Under specific conditions, the RED recombinase expression is induced, enabling the linear DNA fragment to undergo homologous recombination with the target genome, resulting in the knockout or replacement of the target gene (Datsenko & Wanner, 2000; Yu et al., 2000).

Compared to traditional gene knockout methods such as CRISPR-Cas9, RED-mediated homologous recombination offers significant advantages. Firstly, it allows for the introduction of a resistance gene during the knockout process, facilitating the selection of positive clones. Secondly, the RED system does not rely on specific PAM sequences, making it applicable to a broader range of gene editing scenarios. Additionally, due to the reliance on longer homology arms, the RED recombination method provides high specificity and accuracy, ensuring the precise knockout of the target gene (Yu et al., 2000).

We believe that the RED-mediated homologous recombination technique holds significant importance for other teams within the iGEM community. By using this method, teams can perform gene knockouts and replacements with greater flexibility, providing a powerful tool for research and applications in synthetic biology.

In this project, we utilized a temperature-sensitive promoter to control the expression of DpnI, thereby establishing a self-destruction mechanism in the bacterial strain. The mechanism is based on the fact that the temperature-sensitive promoter promotes DpnI expression at 37°C while inhibiting its expression at 30°C or lower. DpnI is a methylation-specific endonuclease that recognizes and cleaves methylated DNA in the E. coli genome. Therefore, at 37°C, the genomic DNA of the strain is cleaved, leading to cell death, while the strain survives at lower temperatures.

Our team proposed utilizing this temperature-sensitive promoter to express the lactose repressor protein (LacI) and link it to the DpnI gene, inducing cell death at temperatures below 30°C. This design enables genome self-destruction at specific temperatures, preventing the leakage of genetic information.

Compared to other kill switches(mechanisms), this design has a unique advantage: by shredding the genetic material, it effectively prevents genetic information from being captured and utilized by other cells. In contrast to other protein-toxicity-based kill switches, this genome-cleavage strategy is more thorough, providing better security for genetic information (Williams et al., 2020).

These technological innovations offer new ideas and tools for the iGEM community and are expected to play an important role in future genetic engineering and biosafety applications.

We designed an innovative protein release system based on the combination of a temperature-sensitive promoter and DpnI. In this system, cells express the target protein at low temperatures and self-destruct at higher temperatures, releasing the expressed enzyme or other target proteins. This method effectively avoids DNA interference and enables more precise protein production and release

Specifically, under low-temperature conditions (30°C or lower), the temperature-sensitive promoter is repressed, preventing the expression of the DpnI gene, allowing E. coli cells to survive and carry out normal protein expression. This allows us to achieve large-scale production of the target protein within the cells.

Once the desired protein expression level is reached, the temperature is raised to 37°C, activating the temperature-sensitive promoter and inducing the expression of DpnI. DpnI is a methylation-specific endonuclease that recognizes and cleaves methylated DNA, leading to the destruction of the genomic DNA. As the DNA is degraded, the cells rapidly undergo self-destruction, releasing the target protein that was previously expressed at low temperatures. During this process, DpnI ensures that the DNA is completely fragmented, thereby preventing contamination of the final protein product during subsequent purification steps.

This temperature-sensitive self-destruction system allows us to achieve the following objectives:

Experimental results demonstrate that through temperature regulation, this system effectively combines protein expression with a self-destruction mechanism, offering high efficiency during protein production and purification. This design provides a novel approach for cell-free protein expression systems, particularly suited for biopharmaceuticals, enzyme production, and applications requiring DNA-free environments.

Additionally, we have uploaded this temperature-sensitive promoter and DpnI-based self-destruction system to the iGEM parts registry under the part number BBa_K5480009. Readers can visit the following link for more details on this part: BBa_K5480009

In our project, we developed an innovative software tool that allows users to encode Chinese text into DNA codon sequences for concealed storage and secure transmission of information. This tool not only enables the conversion of Chinese characters into DNA sequences but also incorporates encoding rules that disguise the information at the genetic level, making it difficult to detect and decode. The software offers several features that could benefit the iGEM community, such as mutation readability analysis and hash value verification to ensure the integrity and accuracy of the data during transmission and storage.

One of the key highlights of this software is its Chinese encoding scheme, which utilizes the logic of the Wubi input method to efficiently convert Chinese characters into DNA sequences. Each Chinese character is represented by 12 base pairs, ensuring compact and high-density data storage. This encoding scheme is not only efficient but also enables the use of codons that form alpha-helices and beta-sheets, disguising the encoded sequences as natural proteins, which enhances data concealment at the biological level.

In addition, the software includes a mutation readability analysis feature that simulates random mutations in the encoded DNA sequences and predicts how they affect the readability of the information. This function helps users understand the robustness of their encoded data against potential mutations. Furthermore, a hash value verification mechanism is integrated to verify the integrity of the data by adding a hash value at the end of the encoded sequence, detecting any potential errors caused by mutations.

Through these innovative features, our software tool offers a novel approach to secure information storage and transmission using DNA, and we are excited to share this tool with the iGEM community. By using this tool, teams can explore new ways to encode and disguise information, providing a powerful method for bioinformatics applications and data security.

• Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences, 97(12), 6640-6645.

• Williams, R., et al. (2020). Engineering biosafety in highly pathogenic bacteria: Synthetic biology-based containment strategies for an age of global pandemics. Journal of Biosafety and Biosecurity, 2(2), 71-78.

• Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G., & Court, D. L. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proceedings of the National Academy of Sciences, 97(11), 5978-5983.