Strain selection

There are also safety considerations incorporated into the design of our project. The final chosen chassis bacteria is E. coli Nissle 1917, which is listed on the white list, classified as biosafety level 1 (BSL-1).

E. coli Nissle 1917 is a non-pathogenic strain of E. coli, commonly used in the treatment of various gastrointestinal disorders, including diarrhea, simple diverticulosis, and ulcerative colitis (UC) ^[3,4]. Extensive research has demonstrated its intestinal anti-inflammatory effects without significant immunotoxic properties.

During the early proof-of-concept phase, we utilized the bacterium Bl21, considering the heavy cost to use Nissle 1917. Bl21 is a non-toxic, protein-inducible expression strain derived from lineage B Escherichia coli, widely employed as a protein expression strain in laboratory settings^[5]. Consequently, our team selected Bl21 as a proof-of-concept strain to assess the feasibility of the genetic circuit.

Kill-switch circuit

1.Reviews of kill-switch circuits

In the field of therapeutic synthetic biology, safety also involves designing measures to prevent engineered cells from harming host cells and environment. Alongside conventional methods like sterilization, the kill-switch circuit has been widely used as the first choice by previous researchers and iGEMers.

The main body of the kill-switch circuit usually comprises two essential components: a sensor and a suicide effector. The sensor detects environmental changes that can activate the suicide effector thus inducing apoptosis. These environmental changes can be generated artificially, such as via the addition of inducers like arabinose or rhamnose. They can also be inherent differences between two distinct environments, such as temperature differences between the body and the external environment, differences in phosphate levels inside and outside blood vessels, pH variances and glucose differences concentration between inside and outside the intestine.

The advantage of adding inducers is that the response can be artificially controlled. However, implementation requires a clear understanding of the metabolism of the inducer in the human body for they may be quickly consumed in the intestine. Typically, this approach is used for emergency when there is a risk of losing control in vivo. Utilizing natural differences between environments, as mentioned earlier, has the advantage of leveraging inherent contrasts. However, it lacks artificial control and is usually employed for suicide when engineered bacteria have been expelled from the body.

In addition to the basic components, sensor and suicide effectors, several other components can be optimized for the kill-switch circuit, including negative feedback components, amplifier components, and quorum sensing components^[6,7,8]. Among these, amplifiers have gained increasing attention, particularly in cases when the concentration of the inducer is low and the initiation strength is insufficient. Amplifiers can enhance the strength through positive feedback, enabling the efficient realization of the suicide effect even with a low concentration of the inducer.

In our project, engineered ECN producing 5-HT were utilized to alleviate constipation, sensing changes in butyrate concentration in the intestine to release 5-HT^[9], aiming to provide long-term preventive effects for individuals prone to constipation. In the four generation of our safety design, which will be mentioned later, we have considered the following concerns:

1.The uncontrolled proliferation of engineered 5-HT ECNs results in excessive production of 5-HT, which disrupts intestinal physiology, even leading to alterations in the microbial composition ^[10].

2.Engineered 5-HT ECNs have the ability to survive and replicate for a certain time outside the body after being excreted in feces, which raises concerns about potential environmental hazards, especially in aquatic environments.

3.The efficacy of engineered 5-HT ECNs is compromised as they are significantly depleted by gastric juices, preventing the desired effect from being achieved.

  

2.Previous Strategies in kill-switch circuits

Previous teams confronted with the same situation have devised several intriguing solutions, each offering their own advantages and disadvantages. In this context, we present a summary of the kill-switch designs developed by teams in 2022 and 2021. Notably, numerous teams have successfully executed proof of concept for their designs, which serves as a valuable reference and inspiration for our work.

(1) Temperature control systems: UFMG_UFV_Brazil_2022 ^[11], NEU_China_A_2018 ^[12]

The temperature control systems leverage the inherent temperature disparity between the internal and external body environments. This enables the engineered bacteria to trigger cell death in cooler external temperatures.

Advantages: It harnesses natural temperature differentials without human intervention.

Disadvantages: Consideration needs to be given to cold temperatures during transportation and the potential escape of bacteria in hot weather. Of course, if enteric engineered bacteria are released into cold water, they can barely escape, even in high summer temperatures.

(2) Oxygen control system: UFMG_UFV_Brazil_2022

The oxygen control system capitalizes on the low oxygen levels in the gut to establish a natural distinction from the external environment. This leads to cell death at higher oxygen concentrations outside the body.

Advantages: It also utilizes natural disparities, and is more stable compared to temperature differences.

Disadvantages: Consideration needs to be given to the low oxygen environment during transportation, as well as the possible liquid environment with low oxygen if expelled out.

(3) Drug Control System: NDNF_China_2021, NYCU_Taipei_2021

The drug control system induces bacterial death by adding inducers such as rhamnose and arabinose. These inducers activate the corresponding inducible promoters, leading to the expression of toxic proteins.

Advantages: The system is man-controlled and utilizes non-toxic and non-hazardous inducers.

Disadvantages: In practical applications, we need to take individual differences in the digestion and metabolism of the inducers into consideration.

(4) Glucose Starvation System: SZU_2021 ^[13]

The glucose starvation system, developed by SZU in 2021, involves the use of glucose starvation receptors. These receptors induce downstream gene expression when glucose is absent. The system takes advantage of the inherent difference in glucose concentration between the gut and external environment to induce bacterial death in vitro.

Advantages: The system relies on inherent differences and offers stability. It has a minimal environmental impact and is easy to set up transportation or preservation conditions (in these conditions, a certain concentration of glucose can be added).

Disadvantages: Additional experimental data is necessary to support the system.

(5) Phosphate sensing system: SZU_2021 ^[14]

The phosphate receptor, also a receptor characterized by SZU 2021, takes advantage of the difference in phosphate concentration inside and outside the blood vessel to prevent engineered bacteria from entering the circulation through minor vascular rupture in the gut under inflammation.

Advantages:The system skillfully solves the problem of preventing the bacteria from entering the bloodstream, a problem less considered by other teams.

Disadvantages: It needs to be supported by more experimental data.

(6) Light induction: SZPT-CHINA_2021 ^[15]

Blue light sensing system to induce bacterial death in natural light.

Advantages: It is easy and stabilized to regulate the system.

Disadvantages: In the shade, when the light intensity is insufficient, it may require human manipulation of blue light to induce death.

(7) Mutation deficiency System: IISER_Kolkata_2021 ^[16]

By inducing mutations in bacteria to strip their ability to synthesize or metabolize a particular substance, bacteria can only survive when the certain substance is artificially added or removed.

Advantages: The system allows for human selection of mutant genes, offering high operability.

Disadvantages: The operation is complex, and it may not be adequately supported by common laboratory techniques.

In conjunction with these approaches mentioned above, the goals for designing the suicide route in our project should include:

1.Automatically activating the suicide program once the engineered bacteria are excreted from the body to prevent environmental contamination.

2.Providing the patient with the ability to control the engineered bacteria, activating the suicide program while the engineered bacteria are still in the patient's intestines.

The former aspect focuses on safeguarding the ecological environment, while the latter emphasizes protecting the patient's health and their right to freely control treatment. The design and iteration of the suicide route in this project will revolve around these two core objectives.

Kill switch design in our project

Guided by human practice, our kill switch went through three iterations, which is described on our page Engineering Success in details

  

Initial design

In our initial design, we firstly considered the need to exterminate engineered bacteria upon their release into the environment and decided to choose the currently maturest temperature control strategy. Bacteria can grow normally in human body at 37℃. However, when the bacteria are discharged out of body and the temperature drops below 30℃, it induces the expression of toxic proteins (such as mazF, ccdB, rel, etc.), leading to bacterial death^[17,18].

However, we identified a defect in our initial design through human practice and experts consultation: the lack of consideration for urgent in vivo containment within the body, which can enhance patients' sense of security, enabling patients to kill and discard engineered bacteria autonomously. Having a self-controlled system will instill a stronger sense of security among individuals.

  

Second-generation design

In response to the concerns regarding the in-vivo safety of bacteria, we conceived the idea of designing an internal containment system by introducing drug control system. We utilized the l-malic acid-sensing switch to induce bacteria death, alongside the rhamnose switch for environmentally exposed death, which constitute a double drug control system.

The double drug control strategy is implemented through the induction of rhamnose and l-malic acid. In an environment lacking rhamnose, the engineered bacteria will not express the tetR, and the ptet promoter activates the expression of the toxic protein mazF, thereby triggering apoptosis, which makes rhamnose a necessary addition for their survival. The l-malic acid switch^[19,20,21] is activated by l-malic acid and initiates the expression of downstream toxic proteins, allowing patients to independently eradicate the bacteria in their bodies by taking l-malic acid.

We assessed the effectiveness of malic acid addition in inhibiting the activity of engineered bacteria and compared the effects of different concentrations of rhamnose on the growth of the engineered bacteria to verify if rhamnose can sustain the viability of the engineered bacteria. Detailed information about our experiment results can be found on our page Results and Experiments.

However, we still found two deficiencies if put into practice. Firstly, l-malic acid also presents in many fruits, which may accidentally trigger the kill-switch after eating abundant fruits. Further investigation is needed to compare the l-malic acid sensing threshold with the concentration of l-malic acid in the intestines after consuming abundant fruits to determine the viability of this strategy.

Secondly, in addition to the engineered bacteria, the intestines harbor a vast population of other microbial communities. Currently, it is uncertain how these communities may affect the concentration of rhamnose. Furthermore, the complex environment of the intestines could potentially lead to rapid depletion of rhamnose to a lower concentration than the threshold required to maintain the activity of the engineered bacteria. One possible solution is to incorporate a positive feedback switch, which would allow even lower concentrations of rhamnose to sustain microbial activity. Moreover, another questionnaire survey carried out by our HPers showed that yogurt as the carrier was more preferred than capsules among the elderly. Considering that products like yogurt demand higher transportation and preservation conditions, we may design a yogurt specialized kill-switch circuit.

  

Third-generation design

Another questionnaire survey targeting the elderly was conducted. The survey specifically compared two delivery methods, yogurt and capsules. The results indicated that the elderly preferred the form of yogurt. We then had a discussion concerning above results with biomedical expert Liao Xiang . He held the points that both yogurt and capsules can be used as carriers for engineered bacteria, but if yogurt is used, the packages need to be designed to withstand stomach acid and transportation issues need to be considered.

Therefore, we explored new kill-switch circuit specialized for yogurt and acid resistant coats^[22] for bacteria, for yogurt can not be made into an enteric form like capsules, which can been found in our Cycle of coats with details and validating data.

Module 1: TET amplification circuit

The safety module utilizes a bistable structure TetR-on system. Upon the administration of TET, the engineered bacteria express the MazF protein, which induces bacterial apoptosis.

The skeleton of this module is originated from Michael B. Elowitz & Stanislas Leibler, 2000^[7].

Module 2: temperature control circuit

We envisioned three possible scenarios for yogurt product carrying our engineered bacteria.

Scenario One: Transportation scenario, where the temperature is below 30℃. In this scenario, Cl⁸⁵⁷ inhibits pR, resulting in no expression of mazE but expression of mazF. However, the addition of yogurt containing rhamnose allows for simultaneous expression of mazE in the engineered bacteria, preventing bacterial death.

Scenario Two: In-vivo scenario, where the temperature is 37℃, Cl⁸⁵⁷ releases the inhibition on pR, leading to the expression of phIF and suppression of downstream mazF expression. At the same time, mazE is expressed. Regardless of whether there is a sufficient concentration of rhamnose to induce mazE, the bacteria can survive.

Scenario Three: Excretion scenario, where the temperature in the sewer system is below 30℃, mazE is not expressed while mazF is expressed. Additionally, rhamnose is diluted, insufficient to induce mazE expression. As a result, the bacteria die.

The skeleton of this module is originated from Wang, et al. 2021.^[23]

Currently, we have only conducted experimental designs for these two modules. Although the framework of the circuits has been well characterized in the literature and stood the test of time, we will strive to complete and refine our validation in the future.

Module 3:Tannin and mucin layer-by-layer coating

Tannin and Ca²⁺ complexation forms a uniform coating on the surface of the probiotic, and then we modified the acid-resistant biomaterial mucin on the outermost layer through the interaction of tannin and glycoprotein. Tannin has strong free radical scavenging and antioxidant abilities, which can improve the survival rate of engineered bacteria. Mucin interacts with intestinal mucous layer through hydrogen bond, disulfide bond and hydrophobicity, thus achieving colonization for a period of time, which has been demonstrated in the study by Yang, et al. ^[22]

The idea of this module is originated from Yang, et al and detailed information about our experiments can been found in our Cycle of coats with details and validating data.

  

Further prospect

Once again, we acknowledged that the third-generation design has certain imperfections:

1. Tetracycline, as an antibiotic, has certain side effects and may have negative impacts on other gut microbiota. Although the designed positive feedback amplification system allows for the induction of engineered bacteria death at very low concentrations of tet, exploring alternative systems is highly necessary for safety considerations.

2. During our review of the previous team's wiki, we discovered that in the case of inflammatory constipation, there may be damaged microvessels in the intestine. Could the engineered bacteria potentially take advantage of this opportunity for invasion? SZU_2021 has already provided a good response to this question.

3. Meanwhile, our PI Changhai Lei raised a question that whether we could construct a Quorum Sensing Module to further ensure the safety by controlling the population density^[8], which would better reflect the preventive role of our engineered bacteria. We did the initial design and conceptualization:

Module ①

When the population density is low, the dominant promoter expresses the downstream genes LuxI and LuxR, which express a small amount of AHL protein and LuxR protein, respectively, and due to the low concentration of AHL protein, it is not enough to bind all LuxR protein. The free LuxR protein cannot induce the luxl promoter to express tetR and bga2 proteins, and can only induce the tetR promoter to express GDH (glutamate dehydrogenase), so it does not lead to bacterial death.

When the population density is high, the dominant promoter increases the expression of the downstream gene Lux, which expresses a large amount of AHL protein. And the high concentration of AHL protein binds to LuxR and forms a dimer. This dimer has the opposite effect to the free LuxR protein, inducing the luxl promoter to express tetR and bga2 proteins, and failing to induce the tetR promoter to express GDH (Glutamate dehydrogenase), while at the same time the induced expression of the tetR protein also inhibits the expression of GDH. And GDH is a protein that must be expressed for cell growth, so inhibition of GDH expression will lead to bacterial death.

Module ②

In normal people, the concentration of butyrate is high, which leads to the high expression of tetR downstream of PLEE. No matter how the population density, tetR’s sufficient inhibition to the promoter PtetR prevents the expression of GDH in the downstream of PtetR. Besides, the effect is more obvious when the population density is high (because tetR can also be produced in the downstream of PluxI promoter), so the bacteria can not proliferate.

In people with constipation, the concentration of butyrate is low, so the amount of tetR expression is also low. If the population density is high, the PluxI promoter will be active, so enough tetR still can be produced, then bga2 expresses but GDH is suppressed. Only when butyrate and population density are both low that makes PtetR active due to insufficient inhibition of tetR production. Then the expression of downstream GDH is promoted and the number of bacteria grows.

The quorum sensing system keeps the population under control and allows the colony to multiply rapidly in certain conditions. An important prerequisite for this system is that the colonies are able to colonize over a period of time rather than passing through the gut. In cycle3, we have designed a mucin and tannin encapsulation with reference to the literature. According to the report, bacteria can colonize for several days after encapsulation. So such an encapsulation can be used in conjunction with a quorum sensing system to achieve colonization and population control simultaneously over a period of time. However, how to further extend the colonization time to better demonstrate the constipation preventive role of our engineered bacteria is one of the directions of our future work.

Parts and materials safety

When constructing the aforementioned kill-switch circuit, we conducted thorough evaluations of the safety of experimental materials and genetic elements. Our priority was to propose strict measures to avoid potential hazards, ensuring the utmost protection for both operators and the environment.

Our basic parts

We designed six basic parts in total this year, integral to our cycle. You can click on the part to see details.

Name	Type	Description
BBa K4656000	Coding	pchA
BBa K4656001	Coding	TDC
BBa K4656002	Coding	TPH
BBa K4656003	Coding	Lrp
BBa K4656004	Promoter	Plee1
BBa K4656005	Promoter	PpchA

Our composite parts

We designed seven composite parts in total this year, which are the backbone of our circuits. You can click on the part to see details.

Name	Type	Description
BBa K4656007	Composite	Plam-TPH-TDC
BBa K4656008	Composite	pchA-PpchA-PLEE1-Cl
BBa K4656009	Composite	Plac-tetR-Ptet-lacI-MazF
BBa K4656010	Composite	PRM-Cl857-PR-phIF-MazE-PphIF-MazF
BBa K4656011	Composite	pRha-tetR-tetO-mazF
BBa K4656012	Composite	pT7-mleR-p_mleS-mazF
BBa K4656013	Composite	pRha-mazE

MazF

MazF is a stable cytotoxic protein encoded by the MazF gene, serving as a ribonucleic acid endonuclease and can be recognized by MazE, which triggers a conformational change of MazF. This conformational change leads to the polymerization of MazE into a hexameric pattern and inhibits the toxicity of the MazF. When MazE is degraded, the MazF is released, resulting in an increase in its concentration and initiating the process of cell death.

The mechanism of action of MazF involves the cleavage of mRNA to prevent translation. As a form of programmed death, it does not inhibit the formation of the bacterial cell wall nor cause lysis of the bacteria. So there is no need to consider the effects of the entry of massive cell contents into the intestine from substantial ruptured dead bacteria.

MazF has been well characterized on BBa_K302033 by iGEM10_Newcastle.

Tannins and mucins

Tannins are substances commonly found in tea and pose no harm to human body in small quantities. Mucins, also known as mucoproteins, are a class of glycoproteins primarily composed of mucopolysaccharides. They have the ability to form gels, making them vital components of gel-like secretions. Importantly, they are non-toxic to both humans and the environment, that is a major reason for choosing them as coating materials^[19].

Rhamnose

Rhamnose (C₆>H₁₂O₅) serves multiple purposes, including determining the permeability of the intestinal tract, acting as a sweetener, producing flavors and fragrances and activating the promoter pRha. Importantly, rhamnose is safe for consumption and pose no harm to the human body or the environment. It serves as a inducer in our circuit pRha-tetR-tetO-mazF and pRha-mazE, which has been described in BBa_K4656011 and BBa_K4656013.

Malic acid

Malic acid, also known as 2-hydroxybutanedioic acid, has two stereoisomers with a special pleasant acidic flavor, mainly used in food and pharmaceutical industries. Among them, L-malic acid is an important ingredient of natural fruit juice, an essential organic acid, a cyclic intermediate of tricarboxylic acid in living organisms, and an ideal low-calorie food additive. Not only that, L-malic acid can also be used for the treatment of liver disease, anemia and other diseases. So it's safe to take it as a inducer in our circuit pT7-mleR-p_mleS-mazF, which has been described on BBa_K4656012.

Tet

Tetracycline is an organic compound extracted from the culture of Streptomyces griseus or dechlorinated from chlortetracycline, with the molecular formula C₂₂H₂₄N₂O₈. They are not very toxic, but side effects are common, such as nausea, vomiting, loss of appetite, secondary infections, and effects on bone and tooth growth. In our circuit, we designed plac-tetR-ptet-lacI to reach a positive feedback, which means only a small amount is needed to initiate downstream expression.

Risk identification and prevention

1.Laboratory Risk Scenario Identification and Response

We have underwent comprehensive laboratory training beforehand and there has been a significant improvement in everyone's awareness of safety and precautions. However, despite adequate preparation, dangers can arise unexpectedly beyond our control, such as machinery malfunctions or accidental mishandling of experiments. Identifying risks and implementing effective planning are crucial steps to ensure a safe working environment.

1.Scalded while using an alcohol lamp, autoclaving, or making agarose gel → Immediately rinse with cold water, soak, and apply scald medication.

2.Cut by a sharp instrument while cutting agarose gel → Immediately clean the wound, disinfect and bandage it to prevent infection, and send it to a doctor for serious injuries.

3.Poisoning by chemical exposure or inhalation → Move outdoors, flush eyes with eye flushes if in eyes, use antidote early and in sufficient quantity, get medical attention immediately.

4.Fire and other large-scale accidents → When the accident is small and you are able to handle it, use appropriate firefighting equipment, emergency sprayers, etc. to handle it urgently after disconnecting the power; when the accident is large, choose a fire escape to evacuate immediately and call the emergency number.

5.Accidental spillage of engineered bacteria or related biologically active substances during work → Immediately decontaminate the operating environment and disinfect operators' hands.

2.Dual-use identification

The rapid advancement of genetic engineering, exemplified by the emergence of recombinant DNA technology, has propelled the growth of synthetic biology. However, any technological breakthrough can potentially become a tool for unscrupulous individuals. The outcomes, whether positive or negative, ultimately rely on the intentions and actions of those who wield it. While the misapplication of technology can indeed pose significant hazards, we firmly believe that we should not be deterred by this inherent risk. Instead, we should approach the field of synthetic biology with unwavering commitment to meticulous risk assessment, allowing us to design and conduct experiments to the best of our abilities.

Risks of misuse

In this project, we did not use the SSBA attenuated gene and we excluded the risks of in-silico experiments (such as bioinformatics approaches , modeling experiments, health impact modeling of toxins, surveillance mechanisms). However, since our engineered bacteria have added virulence factors, which meets a category DURC Category 2^[23], we will conduct a risk assessment on this project.

We have evaluated the risks that may arise from misuse of the project and found that:

①Since our engineered bacteria is E.coli, it can coexist with the human body's own intestinal flora, causing little harm to the human body, and therefore poses little risk in terms of economics or terrorism.

②Because engineered bacteria are difficult to survive after excretion, almost no genetic contamination occurs, so even if it is misused, the risk in public health, agriculture, and the environment is still small.

③There are measures to deal with misuse——the suicide module and quorum sensing module that we give to engineered bacteria.

To sum up, the potential benefits of our project outweigh the risks, and there is no serious risk if it is misused.

Potential risks of the project

E.coli Nissle 1917 is a nonpathogenic strain of E. coli isolated by Alfred Nissle in 1917 that is less capable of causing disease. Furthermore, genetic modification is not expected to alter these characteristics. Our engineered bacteria transformed from it can not only be well integrated into the human intestinal environment, not be affected by pH and other factors, but also play an efficient role in the gut. In addition, the inserts do not cause harm to the recipient organism and do not cause mutations in other genes. According to the above, it is estimated that the likelihood of our engineered bacteria producing multiple products along the genetic route to cause human immune responses is also very low. The quorum sensing module we give to the engineered bacteria allows it to achieve population growth only when both butyrate and bacterial population density are low, so it does not cause overpropagation of the engineered bacteria, nor does it cause serious disturbance of the human gut flora. Based on the characteristics of the final product, the estimated risks involved in this project are low.

Potential hazards of information and technology used in this project

The risk of potential misuse of knowledge, information and technology in this project is minimal. All methods used in this project are basic molecular biology techniques and basic cell culture techniques, including polymerase chain reaction (PCR), agarose gel electrophoresis, plasmid extraction, enzymatic digestion, enzyme conjugation, bacterial transformation, and bacterial liquid-coated plate method. The digital tools used are from the public domain and are widely used in the construction of genetic circuits. Among the bacteria used in this project, the cell chassis E. coli Nissle 1917 is a proven harmless bacterium, and the E. coli BL21 chemoreceptor cell is a commonly used chemoreceptor cell in the laboratory. The project does not develop any new technologies that may pose a risk of misuse, nor will the knowledge, information, techniques, or products resulting from the research be used to harm humans, crops, the environment, the economy, or security. Nor will the misuse of these technologies have serious consequences for public health, agriculture, the environment, the economy, or terrorism.

Recommendations to Ensure Safety

No further preparation of laboratory personnel is required beyond explaining basic biosafety measures to all laboratory members prior to entering the laboratory. In addition, since the sequences used in this project do not pose a hazard or risk of misuse, no special supervision is required for this experiment.

Nonetheless, we remain well prepared. Our PI has been monitoring our progress. And we are also assisting our PI in drafting a mitigation strategy to determine the adequacy of existing biosafety and biosecurity measures. At the same time, we have a long-term plan for the safety of the program, and we regularly (at least annually) review active DURC risk mitigation strategies to assess the validity of experimental results and definitions and to ensure that our safety efforts are as up-to-date as possible.

3.Identification of future application risks

In the future, with good performance and characteristics, engineered bacteria will show good application prospects in medicine and other fields like food, chemical industry, agriculture, sewage treatment and so on. However, even if the enthusiasm in engineering bacteria research is high globally in recent years, due to the immature technology, the engineering bacteria realm is still in the early stage of development, and it is necessary to identify the risks that may be encountered in future applications. The following questions deserve our collective consideration:

1)When engineering bacteria are used to treat diseases in the future, can they always maintain the autonomy and dynamic function of bacteria, play the function of normal bacteria, and not produce toxic by-products or harm the host due to changes in the environment or conditions?

2)After the bacterial drug delivery system is endowed by engineering means, can the engineered bacteria ensure the safe delivery of drugs to the target site without causing the leakage of drugs or other cargoes during transportation?

3)Whether the engineered bacteria will be recognized by the human body as a foreign matter, causing strong allergic reactions and other immune reactions is also worth noting. Also, will the engineered bacteria mutate themselves during application?

4)Will engineered bacteria plant and infect non-target sites in the human body, then pose a threat to human health? After repeated use, does the human body develop a tolerance to the engineered bacteria, like an antibiotic, then affecting the efficacy as a therapeutic agent?

5)Probability of mutation of engineered bacteria and possible effects of mutation?

It can be seen that there are risks for the future application of engineered bacteria that we need to identify as soon as possible. But we believe that, although the application of engineered bacteria in the future is a coexistence of challenges and opportunities, if we can avoid as many risks as possible, or demonstrate wider possible safety, the future of engineered bacteria application will be much brighter. In response to the question 4 and 5, we have a preliminary discussion in the follow-up content.

Discussion on unexpected infection

The presence of small vascular ruptures in the gut may result in the release of 5-HT ECN into the bloodstream.

First and foremost, the primary indications for the product developed in this project are functional constipation caused by reduced intestinal motility and inflammatory bowel disease serves as a relative contraindication for the use of this product. Therefore, it is recommended to thoroughly evaluate the patient's medical history to exclude the possibility of inflammatory bowel disease before considering its utilization.

Furthermore, during the review of previous team wikis, we discovered that another research team had proposed an effective approach to address this issue. This alternative solution involves the use of the phosphate receptor, a receptor characterized by the SZU_2021. By leveraging the difference in phosphate concentration between the inside and outside of blood vessels, this design prevents the entry of engineered bacteria into the circulation through the ruptured blood vessels during inflammation.

Discussion on mutations

In special cases, if mutation occurs in our engineered bacteria, the circumstances may come up:

1. 5-HT synthesis cannot be regulated or terminated. The metabolic module of the engineered bacteria will no longer be constrained and controlled by the sensing module, leading to non-stop 5-HT synthesis in the human intestine^[10].

2. The engineered bacteria cannot start the suicide program. When the engineered bacteria are expelled from the body or inducers are taken, the suicide switch of the engineered bacteria can not be turned on normally, which will lead to the unrestricted proliferation of the engineered bacteria in the body or pollute the environment when expelled out.

Both of scenarios can pose challenges to the safety of engineered bacteria, so we should also be aware of the genetic mutation issue when promoting the Gut-Sweeper program.

For question 1, we can add a detection window for our project to determine whether the metabolic module of the engineered bacteria is normal or not based on the precursor manifestation of 5-HT poisoning. Once abnormal metabolism of the engineered bacteria is detected, the user should seek medical help to alleviate the 5-HT toxicity, and at the same time use appropriate amount of inducers to kill the engineered bacteria.Clinical features of 5-hydroxytryptamine syndrome conclude: severe muscle tonus, autonomic dysfunction and mental status changes, others include fever, myoclonus, hyperreflexia, convulsions and altered state of mind (fidgeting, elevated mood).

Direct treatment according to the severity of the toxicity, include removal of triggers, provision of supportive therapy, control of agitation, use of 5-HT2A antagonists, control of autonomic dysregulation, and control of hyperthermia.

For question 2, as our suicide module has taken into account most of the possible scenarios, will barely result in a situation where the engineered bacteria get out of control and are unable to commit suicide. However, it is necessary to ensure absolute safety, we recommend to sterilize the excreta thoroughly to completely exclude the possibility of contaminating the environment.

Human practice and policy analysis

1.Safety in human Practice

During our extensive human practice, we gathered information on gastrointestinal function, specifically focusing on factors such as the frequency of constipation, appetite status, and fecal characteristics. Once collected, the data was entered into our system and subjected to systematic analysis, which was then presented visually.

To protect the privacy of participants, we adopted a strict anonymization approach. We ensured that all sensitive personal details, including names and individual constipation histories, remained undisclosed.

Additionally, we have reached out to a wide range of constipated individuals residing in different regions with varying dietary habits. We employ a combination of online and offline methods to ensure a diverse dataset, thereby enhancing the reliability and accuracy of our analysis results.

2.Policy analysis

2.1 Policy Analysis of Live Bacterial Drugs

Live bacterial drugs, also referred to as live bacterial preparations or live biopharmaceuticals (LBP), are composed of live microbial preparations derived from normal microorganisms or substances that promote microbial growth. The FDA defines LBPs as containing live microorganisms, such as certain bacteria, which have the potential to prevent, treat, or cure diseases rather than functioning as vaccines. These live bacterial drugs primarily consist of active bacteria but may also include inactive bacteria and their byproducts.

Live bacterial preparations can be categorized into two types: native bacterial preparations and symbiotic bacterial preparations. The strains used in native bacterial preparations are sourced from the indigenous flora found in the human intestinal tract. They complement the existing native bacteria, such as Bifidobacterium and Lactobacillus, by supplementing their numbers and functions. On the other hand, symbiotic bacterial preparations consist of strains that originate from outside the human intestinal tract but can symbiotically interact with the resident intestinal bacteria. They facilitate the growth, reproduction, or provide direct benefits to human bacteria, such as Bacillus subtilis.

Early first-generation probiotics were primarily employed in food and nutritional additives, with Lactobacillus spp. and Bifidobacterium spp. being the most commonly used strains. Currently, live bacterial drugs have not yet received approval from the U.S. FDA for commercial distribution. However, over 20 companies have been actively engaged in specific research and development of live bacterial biopharmaceuticals, with more than 30 projects underway. Notably, significant progress has been made in clinical phase III trials.

Foreign countries have conducted evidence-based evaluations of the clinical effects of microecology-related drugs and have consequently established application guidelines. The Chinese Pharmacopoeia also includes a dedicated section discussing micro-ecological live bacterial products. In this edition, the Pharmacopoeia outlines the essential requirements for micro-ecological products, covering factors such as the product's preparation method and processes that ensure an adequate count of viable microorganisms in the final product.

Although live bacteria drugs have flown into the limelight, genetically modified live bacteria drugs' safety is still to be speculated. We will think carefully about our product positioning and conduct thorough policy analysis in the future to prepare for the next step.

2.2 Measures for the Safety Management of Genetic Engineering

We provided corresponding responses to the entries with high project relevance, among all of which we met the requirements. The full text can be found in SUPPLEMENT pdf3.

Original text of Measures	Responses
Entry 6 According to the degree of potential danger, genetic engineering work shall be divided into four safety levels: safety level Ⅰ, the type of genetic engineering work is not yet dangerous to human health and the ecological environment; safety level Ⅱ, the type of genetic engineering work has a low degree of danger to human health and the ecological environment; safety level Ⅲ, the type of genetic engineering work has a moderate danger to human health and the ecological environment; safety level Ⅳ, the type of genetic engineering work has a high degree of danger to human health and the ecological environment.	Our project belongs to safety level I.Our project has no danger to human health and the ecological environment.
Entry 9 In engaging in experimental research on genetic engineering, safety evaluation shall be conducted on DNA donors, vectors, hosts and genetic engineering bodies. The safety evaluation shall focus on the pathogenicity, carcinogenicity, drug resistance, transferability and ecological and environmental effects of the target genes, vectors, hosts and genetic engineering bodies, as well as determine the levels of biological control and physical control.	We carry out safety evaluation on our project before starting experiment. BL21 is a widely- used and well-established expression strain. ECN belongs to probiotics and abundant have used it as an engineered chassis for the treatment of intestinal diseases. Our laboratory is classified as BSL-II with the ability to contain or deter them.
Entry 12 The use of genetically engineered products shall be subject to biological safety testing and safety evaluation to determine the possible impact of genetically engineered products on public health and the ecological environment.	If put into use in the future, a series of testing and evaluations will be a necessity after the product positioning and carrier form have been clarified.
Entry 13 Units engaging in genetic engineering work shall, according to the applicable nature and safety level of genetic engineering products, make declarations in a categorized and hierarchical manner, and can only proceed with such work after approval. Entry 14 For experimental research on genetic engineering, the work belonging to safety level Ⅰ and Ⅱ shall be approved by the administrative person in charge of the unit; the work belonging to safety level Ⅲ shall be examined by the administrative person in charge of the unit and reported to the relevant administrative department of the State Council for approval; and the work belonging to safety level Ⅳ shall be examined by the relevant administrative department of the State Council and reported to the National Committee on Safety of Genetic Engineering for approval.	Our project belongs to safety level I. The administrative person of institutes and colleges examine our project and PI educated us about experimental safety. We perform experiment under PI’s approval.
Entry 20 Units engaging in genetic engineering work shall, in accordance with the level of safety, formulate appropriate safety measures for the control of waste. Measures shall be taken to inactivate the residual genetic engineering organisms before discharge in order to prevent proliferation and pollution of the environment.	We formulate appropriate safety measures for the control of waste in case of pollution of the environment. Waste disposal at will is forbidden in our laboratory.
Entry 21 Units engaging in genetic engineering work shall formulate contingency measures for the prevention of accidents and include them in the rules for safe operation.	Contingency measures for the prevention of accidents are formulated and put up on our laboratory.Everyone engaging in experiment is educated about these measures.
Entry 24 Units and individuals engaged in genetic engineering work must make safety supervision records in earnest. The safety supervision records shall be kept for a period of not less than ten years for verification.	Safety supervision records are a must in our laboratory.Entry and exit from the laboratory and experimental operations must be documented.
Entry 21 Units engaging in genetic engineering work shall formulate contingency measures for the prevention of accidents and include them in the rules for safe operation.	Contingency measures for the prevention of accidents are formulated and put up on our laboratory.Everyone engaging in experiment is educated about these measures.
Entry 25 Units involved in accidents causing damage to public health or environmental pollution as a result of genetic engineering work must take timely measures to control the expansion of the damage and report to the competent authorities concerned.	We keep in touch with competent authorities and try our best to prevent pollution.