This study is divided into three main experimental components: bacterial transmission, gene privacy, and self-destruction mechanisms. First, we designed experiments to explore bacterial transmission through physical contact, specifically handshakes, where we observed that bacteria could indeed be transmitted, albeit with a decreasing quantity over repeated interactions. Second, to enhance information privacy, we knocked out the guaB gene in the BW25113 strain and introduced a caffeine dependency, ensuring that the engineered bacteria could only survive in caffeine-rich environments. This modification provided a controllable growth mechanism, strengthening the security of the genetic information stored in the bacteria. Finally, the self-destruction mechanism, regulated by the DpnI gene and triggered at higher temperatures, was designed to inhibit bacterial growth, acting as a final layer of protection for information security by ensuring the bacteria are destroyed after transmission. Each of these components plays a critical role in ensuring the overall security and functionality of the system, with bacterial transmission enabling the spread of information, caffeine dependency providing selective growth conditions, and the self-destruction mechanism preventing unauthorized access.

In this experiment, we investigated the potential of bacterial transmission through handshakes. This first part of our study includes two key sections: Encoding and transmission possibility. In the Encoding section, we developed a coding method that embeds information into bacterial DNA using synthetic biology techniques. The goal of this method is to encode complex information securely and ensure that it can be transmitted biologically without being easily deciphered. For the transmission possibility, we smeared bacterial strains onto a hand and then observed their transfer across multiple handshakes to evaluate the effectiveness of bacterial transmission. Figure 1 displays the bacterial distribution in culture dishes at various stages of the experiment. For more details on the Encoding process and the software used for our experiments (click run to Software)



Figure 1:
(00) Initial bacterial smear on the hand.
(01) After first handshake.
(02) After second handshake.
(03) After third handshake.
(04) After fourth handshake.



The results clearly demonstrate that bacteria can be transferred through multiple consecutive handshakes. While the bacterial load decreases with each interaction, the presence of bacteria is still evident after several handshakes, highlighting the efficiency of this transmission method. This illustrates how information, in the form of bacterial cells, can be repeatedly passed between individuals.

The experimental design of this module consists of two main parts: guaB gene knockout and the introduction of the Decaf metabolic pathway. First, we used the Lambda-Red recombination method to construct a guaB gene knockout strain, verifying its impact on bacterial growth through the disruption of the purine synthesis pathway, achieving the goal of preventing growth in standard media. Next, we introduced the Decaf metabolic pathway into the guaB knockout strain, using caffeine as a selective metabolite to ensure that the bacteria can only grow in caffeine-containing environments. These two components together form a highly controllable bacterial information transmission system, enhancing information security and privacy protection.

The purpose of this experiment was to construct the pccdk-up-Kan-down plasmid via the Lambda-Red recombination method, knocking out the guaB gene in the BW25113 strain to investigate the impact of this gene on bacterial growth and phenotype.



Figure 2:
(A) Schematic diagram of the pccdk-up-Kan-down plasmid construction.
(B) PCR amplification results showing guaB upstream fragment, KanR fragment, and guaB downstream fragment.
(C) Enzyme digestion verification showing 2654 bp and 2103 bp fragments, confirming the correct plasmid construction.
(D) Colony PCR showing a 2106 bp band, verifying successful gene knockout.
(E) DNA sequencing results confirming the correct insertion and orientation of the fragments.

Using the Red recombination method, we successfully constructed the pccdk-up-Kan-down plasmid containing the guaB upstream, KanR, and guaB downstream fragments. PCR amplification, colony PCR, enzyme digestion, and DNA sequencing all confirmed the successful knockout of the guaB gene and validated the accuracy of the plasmid construction.

The objective of knocking out the guaB gene was to study its crucial role in bacterial metabolism and growth. The correct construction and validation of the plasmid provide a solid foundation for subsequent functional analyses, allowing us to further investigate the specific role of the guaB gene in physiological processes.



Figure 3:
(A) Cellex pulse cell transfection system, used to introduce the plasmid into the BW25113 strain.
(B) Colony PCR gel electrophoresis results showing a 1662 bp band, confirming successful guaB gene knockout.
(C) Growth curves in LB medium, with blue representing the BW25113 strain and green representing the guaB knockout strain.
(D) Phenotype verification, where the guaB knockout strain does not grow on M9 medium but shows growth on M9 medium supplemented with 0.5 mM xanthine.

After successfully introducing the plasmid into the BW25113 strain using the pulse cell transfection system, colony PCR results confirmed the guaB gene knockout. Growth curve analysis shows that the loss of the guaB gene significantly affects the growth of the strain, which is further supported by phenotype verification. The restoration of growth upon the addition of xanthine suggests the crucial role of the guaB gene in purine biosynthesis.

The purpose of this experiment is to construct a plasmid carrying the Decaf metabolic pathway genes and to verify its accuracy. Through PCR amplification, enzyme digestion verification, and DNA sequencing, we ensured that the target gene fragments were successfully inserted into the plasmid and could be effectively expressed in the host bacteria.



Figure 4:
(A) Plasmid map of the Decaf metabolic pathway genes.
(B) PCR amplification results show the successful amplification of the pYB1s, ndmBCAE, and ndmD fragments.
(C) Enzyme digestion shows a 9155 bp fragment, confirming the correct construction of the plasmid.
(D) Five colonies were selected for colony PCR.
(E) Colony PCR shows a 1767 bp target fragment, demonstrating that the plasmid was successfully transferred into the host bacteria.
(F) DNA sequencing results matched the expected fragments, confirming the correct plasmid construction.

Through PCR amplification, enzyme digestion verification, and DNA sequencing, we successfully constructed a plasmid carrying the Decaf metabolic pathway genes. All verification steps indicated that the target genes were successfully integrated into the plasmid and effectively transferred to the host bacteria. This experiment provides a solid foundation for further studies on the role of the Decaf metabolic pathway in bacterial metabolism.

The purpose of this experiment was to introduce the Decaf metabolic pathway plasmid into the BW-ΔguaB strain, generating the BW-ΔguaB-Decaf strain. This aimed to investigate whether the strain would become dependent on caffeine for growth after the Decaf metabolic pathway was added. By observing the growth curves of the strain under different conditions (normal LB, LB supplemented with xanthine, and LB supplemented with caffeine), we sought to verify the role of the Decaf pathway in bacterial metabolism.



Figure 5:
(A) Under normal LB conditions, the blue curve shows the rapid growth of the original BW25113 strain; the green curve (BW-ΔguaB) and the orange curve (BW-ΔguaB-Decaf) show significantly limited growth.
(B) Under LB conditions with 0.5 mM xanthine, the blue curve shows continuous good growth of the original strain, while the green and orange curves show some recovery of growth in the guaB knockout strains, though not to the level of the original strain.
(C) Under LB conditions with 0.5 mM caffeine, the blue curve still shows a high growth rate, while the green curve shows almost no growth. The orange curve shows a significant recovery in growth, indicating that the Decaf-introduced strain can rely on caffeine for growth.

By introducing the Decaf metabolic pathway into the BW-ΔguaB strain, the experiment demonstrated that the strain’s growth was limited in normal LB medium and LB supplemented with xanthine, falling short of the growth observed in the original strain. However, in LB medium supplemented with caffeine, the strain with the Decaf pathway (BW-ΔguaB-Decaf) showed significant recovery in growth, suggesting that the strain could rely on caffeine for growth through the introduced Decaf pathway.

This demonstrates that the introduction of the Decaf metabolic pathway successfully altered the metabolic process of the strain, making it dependent on caffeine. This provides experimental evidence for the potential use of commonly available caffeine as a metabolic supplement in place of xanthine and showcases the potential of genetic engineering to adjust metabolic pathways.

The purpose of this experiment was to investigate the growth of the strain in media made from common beverages containing different concentrations of caffeine through phenotype verification. The goal was to test whether the strain can rely on caffeine for growth in a high-caffeine environment.



Figure 6: Shows the growth of the strain in media made from different caffeine-containing beverages.



Figure 7: Shows the comparison between the control group (M9 + caffeine and M9 media) and the test group (media made from different beverages) in terms of strain growth.

The results clearly show that the strain exhibited superior growth in high-caffeine beverages (such as pre-workout), while no growth was observed in other beverages with lower caffeine content (such as green tea, jasmine tea, oolong tea, and coffee). This indicates that a high concentration of caffeine is necessary for the strain’s growth.Although coffee contains caffeine, no growth was observed in the media made from brewed coffee. This may be due to insufficient caffeine concentration or the complex chemical composition of the coffee, which may have inhibited the strain’s normal metabolic functions. This suggests that, in addition to caffeine concentration, the complexity of the beverage’s composition may also negatively affect the strain’s growth.Comparing the growth results between the control group (M9 + caffeine) and the different beverage-based media further supports the critical role of caffeine concentration. Pre-workout, with its high caffeine content, was the only test group that supported the strain’s growth. This also suggests that high-caffeine products can be considered as a substitute for laboratory-prepared caffeine solutions when optimizing the strain’s growth conditions.

Overall, the experimental results demonstrate that the strain with the introduced Decaf pathway exhibits caffeine dependency in a high-caffeine environment, while beverages with lower caffeine concentrations or more complex compositions are not suitable for its growth. This provides new insights for further research into the metabolic mechanism of the Decaf pathway and how to use commonly available high-caffeine products to optimize strain growth.

The purpose of this experiment was to construct a plasmid containing the DpnI gene and use temperature regulation to control its expression, thereby achieving a self-destruction mechanism in bacteria to ensure information security. We designed a plasmid regulated by the pR and pL promoters, which activates the DpnI gene under specific temperature conditions, leading to bacterial death. This self-destruction mechanism ensures that the genetic information carried by the bacteria is destroyed under certain conditions, preventing information leakage or misuse. The experimental results demonstrated the effectiveness of this system, laying a foundation for future applications in information protection and biosecurity.



Figure 8:
(A) Plasmid map.
(B) PCR amplification shows the target fragment.
(C) Enzyme digestion verifies the correct construction of the plasmid.
(D) Ten colonies were selected for colony PCR.
(E)Colony PCR shows successful amplification of the target fragment.
(F)DNA sequencing results confirm the accuracy of the inserted fragment.

This experiment successfully constructed a plasmid carrying the DpnI gene, which is regulated by the pR and pL promoters, and includes an ampicillin resistance gene as a selection marker. The results from PCR amplification, enzyme digestion, and DNA sequencing confirmed the correctness of the plasmid construction and the accurate insertion of the fragment.

The primary function of the DpnI gene is to trigger a self-destruction mechanism under specific temperature conditions. When the temperature rises to 37°C, the expression of the DpnI gene is activated, leading to the inability of the host bacteria to grow. This characteristic can be used to control bacterial survival and reproduction under specific conditions. Conversely, at 30°C, the expression of the DpnI gene is inhibited, allowing the bacteria to grow normally. This temperature-dependent gene expression regulation mechanism offers the potential for developing a controllable self-destruction system.

Figure 9:
(A) At 30°C, the expression of the DpnI gene is inhibited, allowing the bacteria to grow normally.
(B) At 37°C, the expression of the DpnI gene is activated, and bacterial growth is severely restricted.

The experimental results validated the effect of DpnI gene expression on bacterial growth under different temperature conditions. At 30°C, DpnI expression is inhibited, allowing the bacteria to grow normally. However, at 37°C, DpnI is activated, leading to a significant inhibition of bacterial growth. This demonstrates the feasibility of a temperature-dependent self-destruction mechanism.

This self-destruction mechanism greatly enhances information security, as it ensures that information encoded in the genome cannot be easily retrieved, while also providing a “self-destruct” feature. Once the temperature changes, the expression of the DpnI gene triggers bacterial self-destruction, ensuring that the information is not disseminated or misused in uncontrolled environments. This gene-based self-destruction mechanism offers an innovative solution for secure information transmission and lays the foundation for future applications in biosecurity and information protection.

Throughout this study, we successfully achieved the key objectives of our project: constructing a controllable bacterial information transmission system. By knocking out the guaB gene and introducing the caffeine-dependent Decaf metabolic pathway, we demonstrated selective bacterial growth based on environmental caffeine levels. Furthermore, the introduction of the temperature-controlled DpnI-based self-destruction mechanism ensured the secure and controlled destruction of encoded information under specific conditions. These results validate the overall design of our project, showcasing a novel and reliable approach for encrypted biological information transmission and protection, providing a solid basis for future advancements in biosecurity and information encryption technologies.