Our suicide switch design plays a significant role in the safety and security of synthetic biology. Especially in the application of probiotics like Lactobacillus plantarum, traditional suicide switches are often not suitable for the human gut environment, and many designs rely on suicide mechanisms that are based on external environmental changes. In contrast, our research innovatively developed an in vivo suicide switch for Lactobacillus plantarum, using the PsppA promoter to precisely control the expression of the LysKB317 lysin gene. This mechanism ensures that under specific conditions, such as in the human gut, Lactobacillus plantarum can self-destruct, thus avoiding long-term colonization and potential ecological risks.
The choice of the PsppA promoter is based on its key role in regulating gene expression in Lactobacillus plantarum, especially in the production of bacteriocins. Our research has shown that the PsppA promoter can be activated by an inducer to control the expression of the downstream target gene LysKB317. LysKB317 is an enzyme that can degrade bacterial cell walls, leading to cell lysis. In our design, the expression of LysKB317 is strictly controlled by the PsppA promoter, which not only ensures the safety of the engineered bacteria but also provides a new safety strategy for synthetic biology.
The PsppA promoter in our system is induced by sakacin P, a compound that is recognized as safe for human consumption. The LysKB317 lysin gene, which is specifically tailored to target Lactobacillus species, poses no risk to human health. Notably, LysKB317 exhibits a high degree of selectivity, effecting lysis solely in Lactobacillus while sparing the beneficial gut microbiota. This precision ensures that the equilibrium of the intestinal microbiome is preserved, thus sustaining the stability of the gut's microbial balance.
Furthermore, our suicide switch design has an important innovative point, that is, a delay control device. Through experimental verification, we found that after the addition of the inducer, Lactobacillus plantarum can continue to release small molecules for about 10 hours. This means that if we use this modified Lactobacillus plantarum as a drug delivery system, it can achieve about 10 hours of drug release in the human body, after which the bacteria will self-destruct, thus avoiding excessive accumulation of the drug and potential side effects.
This innovative suicide switch design not only improves the safety of synthetic biology experiments but also provides new safety guarantees for the application of synthetic biology in the fields of probiotics and drug delivery systems. Our research is pioneering in the field of synthetic biology safety and provides an important reference and guidance for the future development of synthetic biology.
| Name | Number | Type | Length | Description |
| PsppA | BBa_K5238012 | Other | 137bp | The PsppA promoter is a strong inducible promoter that regulates gene expression in Lactobacillus plantarum. |
| PsppA+LysKB317 | BBa_K5238013 | Composite | 1031bp | The PsppA promoter controls the expression of the endosome LysKB317, leading to the programmed cell death of Lactobacillus plantarum. |
| PfdhF-SrpR-PsrpR-LySKB317 | BBa_K5238009 | Composite | 1799bp | The entire system acts as a suicide switch; under intestinal hypoxic conditions, the synthesis of suicide proteins is suppressed, but when exposed to high concentrations of oxygen, the expression of suicide proteins is induced, facilitating the self-destruction of Lactobacillus plantarum through apoptosis. |
| P9-folKE-PfdhF-SrpR-PsrpR-LySKB317 | BBa_K5238015 | Composite | 3149bp | Expression System and Induced Suicide Switch. |
In this composite part, we have selected the PsppA promoter and the lysin LysKB317 to achieve the self-destruction of Lactobacillus plantarum.
The PsppA promoter, derived from the sakacin P gene cluster, plays a role in regulating gene expression in Lactobacillus plantarum, particularly in the production of bacteriocins, which are typically regulated through a secretion-based peptide quorum-sensing mechanism. In the expression vector we constructed, the PsppA promoter works in concert with other regulatory elements to precisely control the expression of target genes, such as LysKB317. This mechanism is of significant importance for the development of novel microbial control strategies.
In relevant literature[ Sørvig, Elisabeth et al. “High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors.” Microbiology (Reading, England) vol. 151,Pt 7 (2005): 2439-2449. doi:10.1099/mic.0.28084-0], researchers have found that swapping different promoters, such as the PsppA and PorfX promoters, affects the production of GusA and PepN. In experimental studies, it was discovered that the PsppA promoter can enhance the production of GusA in two host strains. Therefore, we chose the PsppA promoter from Lactobacillus plantarum to participate in the expression of target genes in the vector, thereby promoting the expression of the downstream target gene LysKB317 and achieving the function of a self-destruction switch.
The core of our self-destruction switch is the lysin gene LysKB317, which originates from the phage genome. LysKB317 is an enzyme that degrades bacterial cell walls, leading to cell lysis. In our design, the expression of LysKB317 is strictly regulated by the inducible promoter PsppA, ensuring the safety of the engineered Lactobacillus plantarum.
In this component, for the first time, we students have utilized the PsppA promoter to regulate the expression of the self-destruction gene LysKB317. In our experiments, we designed a control group (without inducer), an experimental group (with inducer), and a blank group (vector without the self-destruction switch to exclude the possible impact of the inducer itself on bacteria). We verified the effectiveness of the self-destruction switch by plotting growth curves.
In the experiment, initially, we constructed a control strain (L168-pSIP403-PsppA-empty, abbreviated as EP) and a strain expressing a suicide switch (L168-pSIP403-PsppA-LysKB317, abbreviated as LysKB317). Subsequently, we established control groups (EP, EP+sakacin P) and experimental groups (LysKB317, LysKB317+ sakacin P), with the inducer concentration of sakacin P set at 25 ng/ml. We activated the corresponding bacteria, cultured them overnight, and prepared the culture medium under the respective conditions in a 96-well plate. Finally, we inoculated the activated EP and LysKB317 into the corresponding wells at a ratio of 1:50. After completion, we placed the 96-well plate into a microplate reader and adjusted the automatic timing measurement parameters of the reader: 37°C, 10 s of shaking before measurement, medium shaking intensity, wavelength at 600 nm, and a full-plate detection every hour for a continuous monitoring period of 24 hours.
Below are the growth curves we detected. As shown in the figure, EP (blue circles) represents the empty vector control, EP+ (purple squares) represents the empty vector control with inducer, LysKB317 (black triangles) represents the strain with the suicide switch plasmid but without inducer, and LysKB317+ (red inverted triangles) represents the strain with the suicide switch plasmid and with inducer. We continuously monitored the growth of Lactobacillus plantarum over a 24-hour period, reflecting the growth of bacteria in different wells of the 96-well plate through the OD values detected by the microplate reader. The x-axis represents time in hours, with each small division representing an automatic OD value detection by the microplate reader every hour. The y-axis represents the detected OD values. (n=4)
First, we compared the EP (blue circles), which represents the empty vector control, with the EP+ (purple squares), which represents the control with inducer, to eliminate the influence of the inducer on bacterial growth. As observed from the graph, the purple line is slightly lower than the blue line, indicating that the addition of the inducer indeed has a subtle effect on bacterial growth.
Next, we focused on comparing LysKB317 (black triangles), which represents the strain with the suicide switch plasmid but without inducer, with LysKB317+ (red inverted triangles), which represents the strain with the suicide switch plasmid and with inducer. As observed from the graph, the red line is indeed lower than the black line, and the inhibitory effect on the growth of Lactobacillus plantarum L168 by the group with the suicide switch and inducer is greater than the subtle influence caused by the inducer alone. Therefore, this experiment demonstrates that the PsppA+LysKB317 module can suppress the growth of Lactobacillus plantarum L168.
Additionally, from the graph, we can observe that after the addition of the inducer, around the 10th hour, the growth curve of Lactobacillus plantarum L168 begins to gradually slow down, indicating a decrease in growth rate. After the 12th to 13th hour, the red line with the suicide switch and inducer starts to flatten, indicating that Lactobacillus plantarum L168 has entered the stationary phase of growth.. This indicates that the PsppA+LysKB317 suicide switch can inhibit the growth of Lactobacillus plantarum within 12 to 13 hours. Assuming that our Lactobacillus plantarum L168 is administered as a medication to patients with sakacin P inducer, it can control the growth of Lactobacillus plantarum in the gut within about 12 hours, thereby controlling the amount of small molecules expressed and released by Lactobacillus plantarum. This effectively prevents the accumulation of excessive amounts of small molecules in the body, achieving controllable dosage.
The sakacin P-induced promoter PsppA is being used for the first time in iGEM, and LysKB317 is a lysin targeting the cell wall of Lactobacillus plantarum. Theoretically, this design should effectively regulate the growth of Lactobacillus plantarum; this ensures the safety of Lactobacillus plantarum when applied for therapeutic purposes and prevents biological leakage.
This composite part is an anaerobically induced self-destruction switch system, which consists of the following components:
1. The hypoxia-inducible promoter PfdhF regulates the expression of the downstream repressor protein SrpR by sensing the oxygen concentration in the surrounding environment, thereby mimicking the hypoxic conditions within the gut.
2. The repressor protein SrpR, which acts by repressing the downstream P-srpR promoter, thereby inhibiting the expression of downstream genes.
3. The promoter P-srpR, which can be regulated by the repressor protein. If the repressor protein is expressed upstream, the promoter will not regulate the expression of downstream genes.
4. The suicide gene LysKB317: The core of our self-destruction switch is the lysin LysKB317, a novel peptidoglycan hydrolase derived from the phage EcoSau, which can induce bacterial lysis. In our design, the expression of LysKB317 is strictly regulated by an inducible promoter, ensuring that cellular autolysis occurs only in the presence of an inducer.
The entire component serves as a suicide switch, the operational principle of which is delineated as follows: Under anaerobic conditions in the gut, the hypoxia-inducible promoter modulates the expression of the downstream SrpR repressor protein. This protein can bind to the PsrpR promoter, thereby blocking the expression of the bacteriocin in Lactobacillus plantarum, allowing the bacteria to survive normally. In contrast, in the high-oxygen environment outside the gut, the expression of the bacteriocin is not inhibited, enabling the autolysis of Lactobacillus plantarum.
Conclusion
Our self-destruction switch design integrates a sophisticated inducible promoter system with an efficient endolysin gene, ensuring the controllability of bacterial cell lysis. This system enhances biosafety by preventing the accidental spread of genetically modified organisms.
Regrettably, we encountered some difficulties when attempting to link the suicide switch with the target gene folKE, and due to time constraints, we have not yet been able to successfully construct this plasmid and introduce it into Lactobacillus plantarum. Therefore, we regret to report that we have not been able to experimentally verify the functionality of this hypoxia-inducible suicide switch. However, we believe that the design still possesses significant innovation and provides a novel approach for the design of suicide switches in Lactobacillus plantarum. We will complete and validate the functionality of this suicide switch in future experiments.
The composite part integrates our previous self-destruction switch, where the P9 promoter regulates the expression of the target gene folKE, and the self-destruction switch modulates the growth of Lactobacillus plantarum. This part primarily consists of the following components.
1.P9 Promoter
The P9 promoter is a strong promoter in Lactobacillus plantarum, capable of effectively initiating the expression of downstream genes.
2.folKE
Tetrahydrofolate, the target product of folKE, binds to LILRB3 to treat Alzheimer's disease.
3.PfdhF
The anaerobic inducible promoter PfdhF senses the oxygen concentration in the surrounding environment to simulate the low-oxygen environment of the gut and expresses the downstream protein SrpR under low-oxygen conditions.
4.SrpR
The repressor protein SrpR inhibits the downstream P-SrpR promoter, thereby achieving regulation.
5.P-SrpR
The promoter P-SrpR can be regulated by the repressor protein SrpR. If SrpR is expressed upstream, the promoter will not initiate the expression of downstream genes.
6.LysKB317
The core of our self-destruction switch is the endolysin LysKB317, a novel peptidoglycan hydrolase derived from the phage EcoSau. Endolysins are enzymes that degrade bacterial cell walls, leading to cell lysis. In our design, the expression of LysKB317 is strictly regulated by an inducible promoter, ensuring that cellular autolysis occurs only in the presence of an inducer.
Through this composite part, we aim to express the folKE gene to produce tetrahydrofolate, which binds to specific receptors for Alzheimer's disease, potentially leading to the cure of related neurological disorders. Simultaneously, the self-destruction switch ensures the controllability of bacterial cell lysis. We have experimentally verified that this system can effectively express tetrahydrofolate and control the growth of Lactobacillus plantarum, preventing the accidental spread of genetically modified organisms and enhancing biosafety.
In summary, through experimentation, we have successfully overexpressed tetrahydrofolate as a therapeutic drug for Alzheimer's disease (AD); at the same time, we have identified the endolysin LysKB317 specific to Lactobacillus plantarum, which is virtually harmless to humans. This ensures the safety of Lactobacillus plantarum when treating human diseases and avoids biological leakage.
Regrettably, we encountered some difficulties when attempting to link the suicide switch with the target gene folKE, and due to time constraints, we have not yet been able to successfully construct this plasmid and introduce it into Lactobacillus plantarum. Therefore, we regret to report that we have not been able to experimentally verify the functionality of this hypoxia-inducible suicide switch. However, we believe that the design still possesses significant innovation and provides a novel approach for the design of suicide switches in Lactobacillus plantarum. We will complete and validate the functionality of this suicide switch in future experiments.
We work in a neat and safe Lab under professional supervision, which is a Level 2 – moderate containment. The lab possesses standard laboratory equipment, and we carried out all our experiments in proper places. Additionally, instructions and warning signs can be seen everywhere in the lab,every team member have received safety training. We also applied a certain number of dedicated rules to ensure Lab safety.
During experiment, we use a biosafety cabinet to handle biological materials, and only use non-pathogenic strains of low-risk organisms to ensure that no hazardous micro-organisms will flow into the natural environment.
Our experiments are also carried out under professional guidance and supervision——our Principal Investigator, Professor Xingyin Liu, an expert in the department of pathogenic microbiology at Nanjing Medical University, She participated and guided our experiments from design to implementation stage, and join our weekly team meeting to follow up on the experiments. We have fully considered the possible risks of the experiments, and all agreed on Professor Liu's suggestions on corresponding solutions. During the experiment, we can go to our laboratory technician Wenye Huang, Xiaoyi Wang and Xiaoting Luan for help. They are lab members of Xingyin Liu Lab and worked as advisors for our project this year.
In addition, our school, Nanjing Medical University, has a consummate laboratory management system , and we can always turn to the school for help if the risk presented turns out to be beyond our supervisors.
Each of our wet lab members has passed the school's Experiment Safety Assessment and got certification before the iGEM competition.
Besides, we all have laboratory experiences and well familiar with the laboratory safety codes .Furthermore, each time before we carried out an experiment, we would received instruction and training of our PI. In order to use the lab during the summer break (July and August), we filled out the application form and got the consent of the college's laboratory management department.
| During the experiment, we have been following the rules below: |
| Always wear personal protective equipment (PPE) and use appropriate gloves, glasses, and lab coat for each experiment |
| Use laminar flow cabinet to avoid microorganism contamination |
| Disinfect the surface with 70% ethanol before and after using the working table No drinks or food are allowed in the laboratory |
| Implement experimental garbage classification :different bins were used for different kind of waste |
| Label and store flammable and dangerous materials in designated areas |
| The use of an autoclave should be supervised by at least one graduate student Drug-resistant strains are carefully managed to prevent their release into the environment |
