We designed a bacterial therapy that will be applied to the human body and eventually discharged into nature with faeces. To ensure that the engineered bacteria will do no harm to the human body and the environment, we have considered many risks that may occur, such as bacterial off-target, cytotoxic peptide early release, bacterial mutation, environmental pollution and so on. From the selection of the chassis to the confirmation of the drug delivery system, and then to the suicide of the chassis, we constantly prioritize safety and have designed many components to improve it.

1. Chassis
Bifidobacterium longum (BL) ATCC15707 was chosen as the chassis bacterial. BL is a probiotic that is innocuous to the human body and commonly utilized in certain yogurt drinks and live bacterial beverage products. It can promote intestinal health and boost human body's immune system Moreover, it has a tendency to accumulate in the TME and penetrate solid tumors, thereby significantly diminishing the invasion into normal tissues and the detrimental effects. Additionally, BL is a strictly anaerobic microorganism that rapidly succumbs to oxidative stress upon expulsion from the body, which effectively eliminates the possibility of leakage and enhances the safety of our project design. In conclusion, BL is an excellent choice of chassis that offers numerous advantages.

Besides, the chassis bacteria and experimental microorganisms we employ meet the official whitelist requirements of the iGEM competition. To further bolster safety measures, we corresponded with the competition organizing committee by email and obtained a favorable response.

2. Delivery
Capsules that disintegrate solely in the colon have been designed, allowing for the delivery of freeze-dried engineering bacteria powder, which ensures that the bacteria are released only when reaching the colon and prevents their diffusion into other normal tissues.

3. Colonization and targeting
Firstly, the selected chassis, BL, has a tendency to colonize the TME and infiltrate solid tumors, thus limiting the probability of spreading to other normal tissues. This effectively reduces the potential risks which could have been posed by other bacterial treatments. Secondly, we display protein fragments of pilus monomer and tumor-targeting protein fragments on the surface of engineered bacteria to combat CRC cells and pathogenic bacteria Fn, which ensures accurate treatment without adverse effects on other normal tissue cells and the intestinal symbiotic microbiota.

4. Hypoxia response
Once the engineered bacteria enter TME, the hypoxia-responsive promoter responses and initiates the expression of downstream effector genes whereas they are unable to respond in normal tissues with high oxygen content. This ensures that the engineered bacteria can function only in TME.

5. Dual killing
5.1 Human Antibacterial Peptides
The antibacterial and anticancer peptide LL-37 we used in the project is one of the few examples of human-derived antimicrobial peptides that have currently been discovered. Additionally, it demonstrates higher safety compared to other exogenous antibacterial medications.

5.2 Bacteria/tumor-targeting protein fragments
As the antibacterial peptide LL-37 has a wide antibacterial spectrum, we appended bacterial and tumor targeting fragments to both ends of the truncated LL-37 peptide to prevent inadvertent targeting of symbiotic microbiota found in the intestine. Thus, DEH can ensure the mission of dual killing of pathogen bacteria and tumor cells without side effects.

5.3 Condition-responsive cleavage linker
Furthermore, the self-cleaving A&B possess the feature of condition-responsive cleavage. Specifically, it is only cleaved and releases antibacterial peptides in the ion environment of the TME, assuring that normal tissues will not be adversely affected by our treatment system. Furthermore, we plan to enhance our approach by utilizing enzyme cleavage sites that are unique to TME as Linkers.

6. Suicide system

Figure1. Suicide Plasmid

6.1 In vivo
To ensure patients’ safety, a time bomb based on a weak RBS was designed to prevent excessive proliferation of engineering bacteria. Once the engineering bacteria has permeated the tumor microenvironment, the expression of toxin is automatically activated. The toxin accumulation is slow due to the effect of the weak RBS, thereby preventing immediate death of the engineering bacteria. During this time frame, it is possible to engineer bacteria that can release cytotoxic we design, thus accomplishing the mission of dual killing. As time passes, the toxin accumulation will eventually reach a certain threshold, at which the engineered bacteria will be killed under their own action, thereby completing the suicide process in vivo.

Figure2. Suicide in vivo

6.2 In vitro
(1) Exclusively anaerobic
The Chassis bacterial Bifidobacterium longum is exclusively anaerobic. If the engineered bacterial escape the intestinal environment, they would swiftly perish from oxidative stress when exposed to high oxygen air.

Since engineering bacteria may proliferate in hypoxic environments like sewers, we developed a suicide switch that would open after sensing the low-temperature conditions in vitro to mitigate the leakage risks, thus limiting the bacteria number in the environment.

(2) Temperature-responsive kill switch
We have opted to implement the MazEF module in our suicide switch design, and build a library of TA system to benefit future teams in utilizing these components. We have installed a temperature-sensitive RNA switch upstream of the antitoxin mazE to regulate its translation. When the engineered bacteria are exposed to a high-temperature environment (37℃ in vivo), the RNA switch is activated, resulting in normal translation of both the antitoxin and toxin. The antitoxin typically hinders the toxin’s effects, so the genetically modified bacteria can endure. At low-temperature environment(25℃ in vitro), the RNA switch forms a hairpin structure, obstructing the antitoxin’s translation and paving way for regular toxin translation. Thus, the suicide program begins.

Figure3. Suicide in vitro

6.3 Supplement

In the development of the suicide switch in the CPU-CHINA 2023 project, we utilised various components from the TA system and discovered its versatile applications beyond this scope. Therefore, we have created a library for the TA system and provided a concise introduction to it, which briefly arranged and condensed its composition, types, targets, present application scenarios, and techniques to benefit future teams in utilizing these components.