Our project follows the Design-Build-Test-Learn cycle, centered on secure information encoding using genetically modified E. coli. The design is divided into three main modules: first, we created E. coli with a guaB gene knockout, allowing selective growth under xanthine or caffeine. Second, we introduced the DeCaf pathway, enabling the use of caffeine to increase transmission stealth. Third, we implemented a temperature-sensitive self-destruct mechanism to prevent information leakage by degrading DNA under non-ideal conditions. Each module was rigorously tested and improved through iterative feedback.

We designed the knockout of the guaB gene to prevent E. coli from synthesizing GTP and dGTP, thereby restricting the growth of the strain. This design aims to ensure that the engineered strain can only grow under specific conditions (e.g., with the addition of exogenous xanthine), thereby enhancing the security of information transmission. The guaB gene, located in the E. coli genome, encodes guanine nucleotide reductase, a crucial enzyme in the synthesis of GTP and dGTP. We initially constructed a targeting fragment, pccdk-up-kana-down, and then used RED homologous recombination to knock out the guaB gene in E. coli BW25113, replacing it with a resistance gene (KanR) for selection.










We first cultured the BW25113 strain and BW-ΔguaB strain in LB medium, using a microplate reader to measure cell density every hour. The growth curve in LB medium showed that the BW25113 strain grew well, while the BW-ΔguaB knockout strain showed significantly impaired growth, confirming the loss of guaB function. We also cultured the engineered strain in M9 medium with and without xanthine. Colony formation experiments showed that the guaB knockout strain’s growth was significantly inhibited in the absence of xanthine, but normal growth was restored when xanthine was added.

The experimental results validated our design hypothesis: knocking out the guaB gene effectively limits the growth of the strain. This outcome demonstrates that by controlling the availability of exogenous xanthine, selective growth control of the engineered strain can be achieved, thereby enhancing the security of information transmission. Furthermore, the successful application of the RED homologous recombination method highlights the reliability of our gene-editing approach, setting the stage for further modifications in subsequent cycles.

The goal of this design is to engineer a bacterial strain that can only grow in the presence of caffeine, providing a controllable mechanism for both information encryption and biological containment. To achieve this, we introduced the caffeine degradation pathway (DeCaf Pathway) into the guaB knockout strain. Since caffeine is more readily available than xanthine, this modification allows the strain to grow selectively in caffeine-containing environments. The pathway, derived from Pseudomonas putida CBB5, degrades caffeine into xanthine, compensating for the GTP and dGTP synthesis deficiency caused by the guaB knockout. Using synthetic biology methods, we introduced the relevant genes of the DeCaf Pathway (ndm DBCAE) into the BW-ΔguaB strain, constructing a new strain, BW-ΔguaB-DeCaf.





We verified the successful construction through resistance screening and gene expression analysis. The constructed BW-ΔguaB-DeCaf strain was inoculated in LB medium containing either caffeine or xanthine, with BW25113 and BW-ΔguaB strains as controls. The results showed that BW-ΔguaB-DeCaf grew well in caffeine medium, while the BW-ΔguaB strain could not survive. Additionally, we tested the ability of various caffeinated beverages to support the growth of the strain, demonstrating that high-caffeine beverages effectively supported the strain’s growth.



Through this cycle, we confirmed that the introduction of the DeCaf pathway enables the guaB knockout strain to grow specifically in caffeine-containing environments, successfully overcoming the growth deficiency caused by the guaB knockout. In our project, this system achieved the goal of controlling bacterial growth through caffeine, validating our core design concept of using caffeine as a selective growth condition to ensure the security and controllability of information transmission. By verifying the system's effectiveness with naturally occurring caffeine sources (such as treated beverages), we fulfilled the design objectives, demonstrating that this system can selectively grow under specific conditions. This not only met the project's expectations but also provided a reliable solution for biological encryption and controlled information transmission.

To prevent information leakage, we designed a self-destruction mechanism that enables the engineered strain to express DNase under normal cultivation conditions at 37°C, thereby degrading its own DNA and leading to cell death. We chose a temperature-sensitive promoter combined with the DpnI gene to achieve this mechanism. The DpnI gene was cloned into an expression vector under the control of the temperature-sensitive promoter and introduced into the BW-ΔguaB-DeCaf strain, constructing a strain capable of self-destruction at elevated temperatures.



We validated the mechanism through temperature gradient experiments. In the test, the constructed strain was cultured at 30°C and 37°C. The results showed that the strain grew normally at 30°C but could hardly survive at 37°C, indicating the successful activation of the self-destruction mechanism.

The experimental results demonstrate that the designed self-destruction mechanism can be effectively triggered under the specified temperature conditions, ensuring that the engineered strain cannot be misused or leaked after information transmission. This mechanism provides a final safeguard for information security, making the entire information transmission system more robust. Additionally, the success of this cycle highlights the effectiveness of integrating temperature-sensitive elements in synthetic biology designs, offering insights into future strategies for controlled gene expression in various applications.