Inflammatory bowel disease (IBD) is a chronic, non-specific inflammatory reaction mainly manifested in the intestinal mucosa. The disease has a long period of onset and cannot be completely cured. It is prone to repeated attacks and has a potential risk of cancer. Medically, it is mainly divided into three subtypes: Crohn's disease (CD), ulcerative colitis (UC) ,and indeterminate colitis (IC). The diet of patients with IBD is strictly restricted. At the same time, conventional drug treatments only bring limited effects to patients. The side effects, high costs, and long-term treatments that cause material and mental pain also seriously impact the quality of their life. At present, more than 10 million people are suffering from IBD and related diseases in the world, and the number of patients will gradually increase in the future[1](Table.1, Fig.1).

Table.1 Conventional treatments and their disadvantages
Fig.1 The figure shows the rates of IBD among different age groups. IBD of over 100/100 000 rate
was more prevalent among individuals aged over 35 and increased rapidly between the ages of 30 and 69. The
highest prevalence rate was observed in those aged 65–69. Similar tendency was observed in incidence with
highest rate among those aged 50–55. The incidence was observed over 20/100 000 in those aged over 15. The
mortality began to increase rapidly from the age of 60 with rates that were consistently high among those aged over 80.

At present, the main pathological mechanism of inflammatory bowel disease is not very clear. Its pathogenesis may be the result of genetic, environmental, intestinal flora, and other factors. In recent years, the imbalance of intestinal flora has been considered one of the main pathological mechanisms of IBD[2]. The diversification of the pathogenesis of IBD makes its precision treatment effect quite limited[3]. We hope to develop a long-term maintenance treatment based on conservative conventional therapies to relieve patients' discomfort, improve their quality of life for a long time, and even treat patients who do not respond to conventional therapies. We have noticed that there are reports of experimental treatments using probiotics such as lactic acid bacteria and bifidobacteria in combination with traditional medicines and have achieved better results than traditional medicine alone. After investigating in the hospital and listening to the suggestions of different doctors and experts, we decided on a cocktail-style auxiliary maintenance treatment from the perspective of synthetic biology and using modified engineered bacteria to treat IBD caused by the imbalance of intestinal flora from multiple angles. In the investigation, we found many factors related to the disease progression of IBD that could be used as potential therapeutic targets (Table.2). Among these factors, we selected the four most feasible factors (Changes in butyrate production, lipopolysaccharide and Antibacterial Protein LL-37, Superoxide dismutase, and Bilesalthydrolase) as targets of cocktail therapy.

In the investigation, we found many factors related to the disease progression of IBD that could be used as potential therapeutic targets (Table.2). Among these factors, we selected the four most feasible factors (Changes in butyrate production, lipopolysaccharide and Antibacterial Protein LL-37, Superoxide dismutase, and Bilesalthydrolase) as targets of cocktail therapy.

Table.2 Summary of potential therapeutic targets for IBD
Cocktail-style treatment of IBD

Selection of chassis engineering bacteria

According to the difficulty of operation, we have designed three sets of application schemes for chassis engineering bacteria (Fig.2). No matter how the part is designed, we hope it can work in commonly used E. coli engineering bacteria such as DH5α or BL21 (DE3) to ensure that it is usable.

After that, we chose E. coli Nissle 1917 as the primary carrier cell. Nissle 1917 belongs to the category of probiotics. It is non-pathogenic and can colonize. It can enhance the intestinal immune regulation ability to a certain extent and the protective ability of the intestinal epithelial mucus barrier. It can also express microbectin, which can inhibit the growth of other microorganisms such as Salmonella typhimurium and enterohemorrhagic Escherichia coli in the inflamed intestine[31]. It has the potential to treat IBD in itself[32,33]. Because it belongs to Escherichia coli, it has more experimental operability than Lactococcus lactis.

Finally, we hope to be able to complete the characterization in Lactococcus lactis. It belongs to the category of probiotics. The lactic acid bacteria attached to the intestinal mucosa of UC patients are significantly reduced, which may be closely related to the pathogenesis of inflammatory bowel disease[34]. Other studies believe that lactic acid bacteria is associated with the oxidative stress state of the intestine and can inhibit the excessive oxidative stress of the intestine[35]. Lactic acid bacteria is also believed to promote the production of short-chain fatty acids in flora such as Clostridium butyricum[36] and is also relatively operable model strains in genetic engineering.

Fig.2 Selection of chassis engineering bacteria.

Modular cocktail therapy

The concept of synthetic biology fits very well with the cocktail therapy we advocate. With particular emphasis on the interchangeability of promoters and open reading frames, we hope to modularize our therapeutic parts to allow us to freely replace the four therapeutic genes and their expression methods.

Butyric acid production

Butyric acid is the first therapeutic factor we consider. Short-chain fatty acids represented by butyric acid are extremely important substances for maintaining colon health. They are the main energy source for colonic epithelial mucosal cells. They can promote the repair of intestinal epithelial cells, strengthen the intestinal mucosal barrier, and inhibit NF-κB and interferon- γ and many other inflammatory pathways to slow down the inflammatory response. It also has high safety and tolerable doses and is considered an ideal drug for IBD treatment[37]. It has been reported that in the intestine of patients with IBD, the proportion of butyric acid-producing bacteria is reduced, and the content of short-chain fatty acids (SCFA) in the intestine is low[38]. However, butyric acid cannot be directly administered orally. It can only be administered by enema or embedded in enteric-coated capsules. However, the efficacy will be limited by factors such as the method of administration, drug concentration, and maintenance time[37]. We believe that through the proper colonization of engineered microorganisms, continuous long-term drug delivery can be expected, and other auxiliary functions can be added to help restore the balance of intestinal flora.

In the fatty acid synthesis pathway, the thioesterase family plays the role of breaking the extended cycle of fatty acids and releasing free fatty acids. Different thioesterases have different degrees of fatty acid chain length specificity. We selected the thioesterase from Bacteroides polymorpha[39,40] and transferred the expression vector plasmid containing its gene into three engineered bacteria we envisioned for expression (Fig.3). In this way, the engineered bacteria can help repair the intestinal environment and achieve the purpose of the long-term release of appropriate butyric acid.

Fig.3 Butyric acid expression element and its mechanism of action.

We verified the successful transformation of the expression vector and proved the correct expression of the protein by protein electrophoresis and affinity chromatography. In addition, we used liquid chromatography to directly measure the butyric acid production of engineered bacteria to verify the feasibility of this element.

The literature pointed out that several cytokines with pro-inflammatory activity, including IL-1, IL-6, IL-8, IL-12, and TNF-α, are up-regulated in inflammatory bowel disease. These indicators play a key role in the clinical and immunopathological manifestations of IBD[41-44]. At the same time, clinical studies have performed ELISA analysis of cytokine levels in the serum of healthy people, UC patients, and CD patients. The results indicate that UC patients have the anti-inflammatory cytokine IL-10 was also significantly higher than that of the healthy control group[45].

Macrophages play a significant role in the pathogenesis of IBD. When a pathogen invades the intestine, the pathogen can pass through the damaged intestinal epithelial cell barrier and stimulate the inner defense cells in the epithelium, especially macrophages. After being stimulated, they will produce pro-inflammatory cytokines and then release IL -1, IL-6, IL-18, TGF-β, and TNF-α, and these cytokines directly or indirectly affect intestinal epithelial cells, causing these cells to damage or necrosis, and promote the occurrence and development of IBD (Fig.4)[46].

Fig.4 Pathological effects of different pro-inflammatory cytokines in the pathogenesis of IBD.

THP-1 is a human leukemia mononuclear cell line that has been widely used to study the functions, mechanisms, signal pathways, nutrition, and drug transport of monocytes/macrophages. THP-1 can be induced to differentiate into macrophages M0 by phorbol ester (PMA) and then induce M1 polarization through lipopolysaccharide(LPS) and IFN-γ, releasing cytokines such as TNF-α and IL-6, becoming typical Model of inflammation. At the same time, the induction of IL-4, IL-13, and macrophage colony-stimulating factor (M-CSF) can achieve M2 polarization and make it secrete inhibitory cytokines such as TGF-β and IL-10. This is similar to the process of tissue repair and reconstruction in the later stage of inflammation[47].

Therefore, to further verify whether the components we constructed can effectively alleviate inflammatory symptoms, we also used the THP-1 cell line with and without LPS to interact with the engineered bacteria. Among them, LPS will increase the production of IL-6, IL-8, and TNF-α pro-inflammatory factors in differentiated THP-1 cells[48]. Afterward, a specific and highly sensitive enzyme-linked immunosorbent assay (ELISA) was used to detect the contents of interleukin-6 and interleukin-10 in cells and analyze their relationship with changes to test whether they have the effect of slow-releasing inflammation-related factors.

We also hope to transplant the engineered bacteria into the colon of IBD model mice induced by dextran sulfate sodium salt (DSS) to simulate treatment. At the same time, metagenomic sequencing will be performed on the feces of the mice before and after transplantation to verify whether the flora in the intestines of the mice was actively restored. At the same time, it is also necessary to evaluate the weight of the mice, observe the section of their colon, and evaluate the effectiveness from the aspects of colon inflammation score, colon length, serum IL-1β, IL-18, IL-33, and other inflammatory factors.

However, due to accidents such as the covid-19 epidemic and the degradation of engineered bacteria encountered during the project's progress, we cannot perform experimental animal characterization and more designed experiments.

Against lipopolysaccharide

Lipopolysaccharides are also involved in disease progression. In UC patients, the intestinal flora is unbalanced, the mucosal barrier function and permeability are changed, and Gram-negative bacteria translocate and enter the lymphatic and blood system. Its lipopolysaccharide stimulates the immune system and binds to Toll-like receptor 4, activating myeloid differentiation factor-dependent pathways. MyD88-dependent pathway recruits and activates the downstream tumor necrosis factor receptor-related factor 6 (TRAF6) through intracellular signaling molecules. The activated TRAF6 further activates the NF-κB signaling pathway, causing inflammation. The human body is very sensitive to bacterial endotoxins. When a large amount of endotoxin enters the blood, endotoxemia can occur. Failure to properly regulate the TLR pathway may lead to common chronic inflammatory diseases. Exaggerated response to LPS stimulation, especially dangerous high levels of cytokines and inflammatory mediators, can lead to more critical, systemic inflammatory response syndrome[49].

LL-37 is an antibacterial peptide secreted by human immune cells, inhibiting harmful bacteria such as Helicobacter pylori and Staphylococcus aureus. The concentration of this peptide (1-5 μg/ml) usually found on the mucosal surface of adults is not enough to directly kill bacteria but participates in immune regulation and protects the human body from endotoxemia/septicemia[50]. Under extremely low concentrations (≤1 μg/ml) and physiological salt conditions in the body, LL-37 has powerful anti-endotoxin properties (Fig.5)[49], which can significantly inhibit the specific pro-inflammatory upregulation of NF-κB Gene expression. Past studies believed that the treatment of LL37 also significantly changed the bacterial species and abundance of the intestinal flora of mice, revealing its important influence on the intestinal flora[51].

Fig.5 LL37 resists LPS mechanism and LL37 expression elements.

Therefore LL37 will be a very potential therapeutic peptide. We hope that the engineered bacteria will directly synthesize LL37 as a therapeutic element, and use an exocrine vector for exocrine expression as a supplementary substance to participate in immune regulation and targeted suppression of LPS. We transformed the plasmid containing LL37 exocrine expression into engineered bacteria and performed special protein electrophoresis to verify the successful expression. We pay more attention to cell-level verification in this section. Therefore, to verify whether it can effectively alleviate inflammation symptoms, we used THP-1 cell line with and without LPS added to interact with engineered bacteria to detect the content of interleukin-6 and interleukin-10 to test whether it has The effect of slow-release inflammation-related factors.

The concentration of LL37 determines its effect. We hope to transplant the engineered bacteria into the colon of DSS-induced IBD model mice to simulate treatment, determine the colonization number of mice colonic flora and determine the concentration of LL37 in the colon mucosa of mice after treatment. This will prove what kind of therapeutic effect the concentration of LL37 has on mice. We will also perform metagenomic sequencing analysis on mice feces before and after treatment to verify whether the flora in their intestines has recovered as expected. At the same time, it is also necessary to evaluate the weight of the mice, observe the section of their colon, and evaluate the effectiveness from the aspects of colon inflammation score, colon length, serum TNF-α, and other inflammatory factors.

However, due to accidents such as the covid-19 epidemic and the degradation of engineered bacteria encountered during the project's progress, we cannot perform experimental animal characterization and more designed experiments.

Superoxide dismutase

Excessive reactive oxygen metabolites play a role in mediating IBD intestinal damage. They damage lipids, proteins, nucleic acids, destroy the integrity of epithelial cells and cause subsequent fluid and electrolyte loss. They are also involved in various intracellular signaling pathways. They regulate the expression of various inflammatory cytokines, adhesion molecules, and enzymes by activating redox-sensitive transcription factors, thereby promoting the continuation of the inflammatory process. The intestinal mucosa has a complex antioxidant system, in which superoxide dismutase (SOD) is the initial enzyme for the operation of the entire system. However, this system has been destroyed in IBD patients[52, 53]. We would naturally think of increasing the amount of superoxide dismutase enzyme in the patient's intestinal tract. However, the half-life of superoxide dismutase is very short, and it cannot be directly administered for treatment and requires carrier delivery.

Past studies have shown that only overexpression of heterologous SOD by engineered bacteria can significantly reduce the peroxidation in the inflamed colon of IBD model mice [54]. Based on this, we hope to use a heterologous exocrine expression system to express superoxide dismutase (Fig.6), to achieve long-term stable therapeutic effects. We transformed the plasmid containing superoxide dismutase exocrine expression into engineered bacteria and performed electrophoresis and Western-Blot respectively at the gene and protein level to verify its successful expression. We also perform enzyme activity assays on cell contents and culture supernatants. For intracellular superoxide dismutase, we will determine its enzyme activity-time curve and enzyme activity-temperature curve and verify it at the level of enzymology.

Fig.6 Superoxide dismutase (SOD) down-regulates superoxide (ROS) mechanism and SOD expression element.

Furthermore, to verify whether it can effectively alleviate inflammatory symptoms, we used THP-1 cell line with and without LPS added to interact with engineered bacteria to detect the content of interleukin-6 and interleukin-10 to test whether it is It has the effect of slow-releasing inflammation-related factors.

We also hope to transplant engineered bacteria into the colon of DSS-induced IBD model mice to simulate treatment and perform a metagenomic analysis of the feces of the mice before and after transplantation to verify whether the intestinal flora is actively restored. At the same time, it is also necessary to assess the weight of the mice and slice their colon to observe the degree of macroscopic damage and the length of the colon, to score by nitrotyrosine immunostaining, and to measure the activity of myeloperoxidase.

However, due to accidents such as the covid-19 epidemic and the degradation of engineered bacteria encountered during the project's progress, we cannot perform experimental animal characterization and more designed experiments.

Bile acid hydrolase

Bile salt hydrolase (BSH) is responsible for the hydrolysis of combined bile salts to produce free bile acids and amino acids. The BSH activity in the gastrointestinal tract mainly comes from Firmicutes (30%), Bacteroides (14.4%), and Actinomycetes (8.9%). It is suggested that the changes in the abundance of BSH gene caused by the changes in the number of Bacteroides and Firmicutes in the intestinal tract of UC patients may be the basic cause of changes in bile acid metabolism[55]. Impaired microbial enzyme activity in IBD patients can lead to impaired bile acid metabolism, characterized by the inability of bile salts to be hydrolyzed, converted, and desulfurized. The BSH activity of microorganisms removes the coupling of glycine or taurine molecules to produce unbound bile acids [56]. Abnormal bile salt metabolism in the intestinal lumen can enhance the inflammatory response of the intestinal epithelium, thereby worsening IBD (Fig.7).

Fig.7 Mechanism of bile acid hydrolysis by bile hydrolase and BSH expression element of bile hydrolase

Ingestion of probiotics with BSH activity can promote the biotransformation of bile salts in the intestine, thereby alleviating the inflammatory response caused by high-dose bile salts in the intestine. As shown in Figure 7d, we hope to express the gene encoding the well-characterized BSH enzyme heterologously with engineered bacteria and make it settle in the intestine, and continuously deliver high levels of enzyme activity locally to supplement the lack of BSH gene abundance caused by IBD. At the gene and protein level, we performed electrophoresis and WB to verify its successful expression. We also measured the enzyme activity of the cell content and determined its enzyme activity-time curve, and verified it at the level of enzymology.

Furthermore, to verify whether it can effectively alleviate inflammatory symptoms, we used THP-1 cell line with and without LPS added to interact with engineered bacteria to detect interleukin-6 and interleukin-10 content.

At the same time, we hope to transplant the engineered bacteria into the colon of IBD model mice induced by DSS to simulate the treatment. We plan to determine the colonization of the colonic flora of mice and total serum cholesterol, low-density lipoprotein cholesterol, triglycerides, etc., liver cholesterol content, feces cholesterol, bile acid, and short-chain fatty acid content. This can help us explore the ability of bile salt hydrolase to interfere with abnormal bile acid metabolism in IBD model mice.

However, due to accidents such as the covid-19 epidemic and the degradation of engineered bacteria encountered during the project's progress, we cannot perform experimental animal characterization and more designed experiments.

Acid inducible

There are not only four options for therapeutic genes, but we also considered two expression methods in order to carry out the standardized replacement of promoters.

In the intestine, our engineered bacteria cannot use isopropyl-beta-D-thiogalactopyranoside (IPTG) to induce an expression system. First of all, iptg has cytotoxicity. In addition, food contains a lot of glucose and lactose. The high content of the former in the stomach and intestines will have an inhibitory effect on this expression system, while the latter will be digested by the human body into galactose and glucose, making the iptg induction system difficult to regulate.

We first chose constitutive expression using the most common strong constitutive promoter P J23100 (Fig.8). Although the prediction model shows that the efficiency of the promoter is much lower than that of the inducible expression system, it can meet the long-term and reasonable demand for drug delivery, and it also has stable and reliable protein expression results in the experiment.

Fig.8 Schematic diagram of constitutive expression.

However, the constitutive expression means that cells express almost all their lives, which will have a certain impact on their growth and colonization. In addition, there is an accumulation of acetic acid and lactic acid in the colon of IBD patients, and the environment is acidic. In contrast, the end of the colon of normal people has a pH of 7.1-7.5. The pH of UC patients is basically lower than 5.7, while the pH of CD patients is also around 5.2. [57]. Therefore, the acid promoter p170 will meet this application scenario and achieve an acid response to the environment. It is a commonly used ph-sensitive promoter in the food-grade expression of lactococcus lactis. It is highly expressed when ph=5.5 and is low or not expressed when ph=7.0 (Fig.9)[58]. We refer to the PrcfB promoter of the 2019 SZPT-China team to achieve the regulatory function we need. It is an improved version of P170 and has a higher expression efficiency.

Fig.9 Expression intensity of acid promoter P170 at different pH values.

The response of this promoter involves a set of endogenous response systems of lactic acid bacteria, which involves a key trans-acting factor RcfB, which needs to be combined with lactic acid or generalized acid to cover the binding site upstream P170 and initiate the activity of P170[59]. This protein is endogenous to lactic acid bacteria, and if we want E. coli to use this system, we tried to transform a plasmid vector expressing the gene into E. coli additionally and use a constitutive promoter to control its expression, hoping to be in E. coli On the application.

We will apply this promoter to four therapeutic elements (Fig.10). Although many teams have verified the function of the promoter, we still use the superoxide dismutase gene to verify that the promoter can adapt to our application scenarios. We will culture the engineered bacteria in media with different pH levels to the same concentration and then test their intracellular superoxide dismutase activity to prove that the promoter is effective.

Fig.10 Principle of acid sensing.

The Promising Cocktail of Therapies


The original cocktail therapy refers to the combined use of three or more antiviral drugs to treat AIDS. It highlights the combined use of multiple factors and multiple angles of multiple drugs. This is where we think about treating IBD. Considering only the two promoter schemes and four therapeutic modules we have proposed, we can already provide eight combinations of individual therapeutic elements, needless to say the potential therapeutic targets we have found and recognized by the academic community.

In the end, we put forward the concept of "treatment closed loop" so that we can track each patient who uses our treatment engineered bacteria and cooperate in-depth with hospitals, biopharmaceutical factories, and bioinformatics analysis companies to track and judge their health status promptly. They provide probiotic medicines that best fit the actual situation.

Specifically, we plan to provide patients and hospitals with our self-developed stool collection device. For patients with serious diseases that require hospitalization or hospital care, diagnosis and treatment will be performed in the hospital, and stool sampling and collection will be completed. Moreover, those patients whose diseases enter the sustained-release period can also collect them at home by themselves. We will collect stool samples from patients and perform metagenomic sequencing to analyze the composition and even dynamic changes of the intestinal flora of patients. Based on this, we will use each treatment element in proportion to each other according to the patient's specific conditions. For example, gene expression intensity can be controlled by changing the proportion of the bacterial population that carries each therapeutic element or using different promoters. According to this ratio, we will produce engineered bacteria drugs and provide patients with the most suitable microbiota adjuvants for their conditions. We will also formulate a diet plan based on the patient's specific situation and ask him to record his daily diet to predict changes in his intestinal flora in advance. Until the next patient's stool sample is received, we will perform analysis and testing again. The entire treatment progress is carried out in this closed-loop, and we believe this will greatly promote the application and development of gene therapy in precision medicine.

Safe treatment of IBD

Cocktail therapy treatment of IBD involves the colonization of engineered bacteria into the human colon. Therefore, the safety of the engineered bacteria itself must be considered, and the safety of resistance and suicide must be ensured.

Resistance selection

The abuse of antibiotics globally has become a serious problem. In order to prevent the additional introduction of engineered bacteria containing antibiotic resistance genes to colonize the digestive tract, and considering the risk of the transfer of antibiotic resistance genes between species, we cannot use antibiotic resistance such as erythromycin, ampicillin and kanamycin as a filter condition. We hope to use heat shock protein and triclosan resistance gene as a dual screening system.

Heat shock protein is a type of heat stress protein that exists widely. When cells are exposed to high temperatures, they will be stimulated by heat to synthesize such proteins to protect themselves and have molecular chaperone activity. It allows cells to acquire the ability to withstand harsh living conditions such as high temperature, acidic environment, ethanol stress, and hydrogen peroxide stress[60]. We can use a temperature of 55°C or even higher for thermal screening. Cells that do not express the protein will not survive for a long time under this condition or will survive very poorly. Moreover, heat shock protein can assist engineering bacteria to better colonize in the acidic colon environment rich in reactive oxygen species, which is in line with our application scenario. Triclosan is a safe and broad-spectrum antibacterial substance certified by the FDA. It is widely used in daily chemicals such as soap and toothpaste. The presence of such substances has also been detected in human breast milk and urine. We endow the engineered bacteria with triclosan resistance by expressing its resistance gene and achieving the screening effect. In order to prevent each screening method from having its own limitations, we integrate these two resistance elements together (Fig.11), to do double resistance screening to ensure efficiency. We also hope that our ideas can be verified in subsequent experiments.

Fig.11 Comic drawing of hsp and FabV expression elements.

For the expression of heat shock protein, we first verify the expression of the protein by protein electrophoresis. After that, we set up different growth stress conditions such as high temperature, ethanol, hydrogen peroxide, and set up long-term cultivation of engineered bacteria after the platform growth period, and observed their survival rate to verify the role of heat shock protein. For the triclosan resistance gene expression, we first verified the expression of the gene by protein electrophoresis. Then set up different concentration gradients for long-term plate culture, count and observe the survival rate of engineered bacteria, and explore the lowest concentration that the system can efficiently screen in different chassis bacteria.

Due to practical time issues, we have not verified these two resistance screening methods on Lactococcus lactis.

Suicide switch


We first consider the excretion of engineered bacteria into the external environment. We noticed a difference in glucose concentration between the human intestine and the external environment, so we considered choosing a promoter PT-αcrp that can respond to changes in glucose concentration. We do not want any bacterial contents to escape when the cells die to avoid any potential risks. Therefore, we chose to use the mazF gene. Encoding an endoribonuclease that can cleave RNA at the ACA site and cause the death of microorganisms, it can mediate cell suicide without causing bacterial cell lysis[61]. The promoter PT-αcr controls the following expression of the suicide gene mazF gene (Fig.12). When the glucose concentration is low, it will initiate the expression of suicide genes to achieve the suicidal effect. We will use different glucose concentrations to induce its expression and observe the survival rate of engineered bacteria.

Fig.12 Glucose concentration sensitive kill switch.

In addition, we still worry that any bacteria will enter the local blood vessels through the damaged intestinal epithelium of the patient. We have noticed a difference in phosphate concentration in the blood vessels and the intestine, and the phosphate concentration in the blood is significantly higher than that in the colon[62]. We use a phosphate-sensitive promoter, using E. coli's endogenous phosphate-sensitive trans-acting factor, activating the promoter at low phosphate concentrations and inhibiting the promoter at high phosphate concentrations. We refer to the XMU-China team’s suicide switch design in 2020 and designed a conversion system to reverse gene expression. In other words,the suicide gene expression will be inhibited at low phosphate concentration and will be promoted at high phosphate concentration. When the PhoB promoter is turned on for expression, the expressed tetR protein will inhibit the activity of the pTet promoter from inhibiting subsequent suicide gene expression (Fig.13). The engineered bacteria will be killed once they enter the high phosphate environment in the blood.

Fig.13 Phosphate-sensitive kill swtich.

[1] Kaplan, G.G., Windsor, J.W. The four epidemiological stages in the global evolution of inflammatory bowel disease. Nat Rev Gastroenterol Hepatol 18, 56–66 (2021).

[2]Yue B, Luo X, Yu Z, Mani S, Wang Z, Dou W. Inflammatory Bowel Disease: A Potential Result from the Collusion between Gut Microbiota and Mucosal Immune System. Microorganisms. 2019;7(10):440. Published 2019 Oct 11. doi:10.3390/microorganisms7100440
[3] 陈白莉 , 钱家鸣 , 吴开春 , 等 . 英夫利西治疗活动性溃疡性结 肠炎疗效与安全性的临床研究 [ J]. 中华炎性肠病杂志 ( 中英 文 ), 2017, 1(1):20-23
[4] Levitt M D, Furne J, Springfield J, et al. Detoxification of hydrogen sulfide and methanethiol in the cecal mucosa[J]. The Journal of clinical investigation, 1999,104(8):1107-1114.
[5] Santhanam S, Venkatraman A, Ramakrishna B S. Impairment of mitochondrial acetoacetyl CoA thiolase activity in the colonic mucosa of patients with ulcerative colitis[J]. Gut, 2007,56(11):1543-1549.
[6] Bai A, Lu N, Guo Y, et al. All-trans retinoic acid down-regulates inflammatory responses by shifting the Treg/Th17 profile in human ulcerative and murine colitis[J]. Journal of leukocyte biology, 2009,86(4):959-969.
[7] Xiao S, Jin H, Korn T, et al. Retinoic acid increases Foxp3+ regulatory T cells and inhibits development of Th17 cells by enhancing TGF-beta-driven Smad3 signaling and inhibiting IL-6 and IL-23 receptor expression[J]. J Immunol, 2008,181(4):2277-2284.
[8] Shimoda M, Horiuchi K, Sasaki A, et al. Epithelial Cell-Derived a Disintegrin and Metalloproteinase-17 Confers Resistance to Colonic Inflammation Through EGFR Activation[J]. EBioMedicine, 2016,5:114-124.
[9] Uchiyama K, Naito Y, Takagi T, Mizushima K, Hayashi N, Handa O, Ishikawa T, Yagi N, Kokura S, Yoshikawa T. FGF19 protects colonic epithelial cells against hydrogen peroxide. Digestion. 2011;83(3):180-3. doi: 10.1159/000321809. Epub 2011 Jan 21. PMID: 21266813.
[10] Laserna-Mendieta E J, Clooney A G, Carretero-Gomez J F, et al. Determinants of Reduced Genetic Capacity for Butyrate Synthesis by the Gut Microbiome in Crohn’s Disease and Ulcerative Colitis[J]. Journal of Crohn's and Colitis, 2018,12(2):204-216.
[11] Liu Y, Peng J, Sun T, et al. Epithelial EZH2 serves as an epigenetic determinant in experimental colitis by inhibiting TNFalpha-mediated inflammation and apoptosis[J]. Proc Natl Acad Sci U S A, 2017,114(19):E3796-E3805.
[12] Ho G, Aird R E, Liu B, et al. MDR1 deficiency impairs mitochondrial homeostasis and promotes intestinal inflammation[J]. Mucosal Immunology, 2018,11(1):120-130.
[13] Li L, Wan G, Han B, et al. Echinacoside alleviated LPS-induced cell apoptosis and inflammation in rat intestine epithelial cells by inhibiting the mTOR/STAT3 pathway[J]. Biomedicine & Pharmacotherapy, 2018,104:622-628.
[14] Sancho-Vaello E, Gil-Carton D, François P, et al. The structure of the antimicrobial human cathelicidin LL-37 shows oligomerization and channel formation in the presence of membrane mimics[J]. Scientific Reports, 2020,10(1)
[15] Zhang H, Zheng Y, Pan Y, et al. A mutation that blocks integrin α4β7 activation prevents adaptive immune-mediated colitis without increasing susceptibility to innate colitis[J]. BMC Biology, 2020,18(1).
[16] Motta J, Palese S, Giorgio C, et al. Increased Mucosal Thrombin is Associated with Crohn’s Disease and Causes Inflammatory Damage through Protease-activated Receptors Activation[J]. Journal of Crohn's and Colitis, 2021,15(5):787-799.
[17] Kobayashi K S. Nod2-Dependent Regulation of Innate and Adaptive Immunity in the Intestinal Tract[J]. Science, 2005,307(5710):731-734.
[18] Okumura R, Kurakawa T, Nakano T, et al. Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia[J]. Nature, 2016,532(7597):117-121.
[19] Hanaei S, Sadr M, Rezaei A, et al. Association of NLRP3 single nucleotide polymorphisms with ulcerative colitis: A case-control study[J]. Clinics and Research in Hepatology and Gastroenterology, 2018,42(3):269-275.
[20] Owyang A M, Zaph C, Wilson E H, et al. Interleukin 25 regulates type 2 cytokine-dependent immunity and limits chronic inflammation in the gastrointestinal tract[J]. Journal of Experimental Medicine, 2006,203(4):843-849.
[21] Yang X O, Chang S H, Park H, et al. Regulation of inflammatory responses by IL-17F[J]. Journal of Experimental Medicine, 2008,205(5):1063-1075.
[22] Zhu L, Han J, Li L, et al. Claudin Family Participates in the Pathogenesis of Inflammatory Bowel Diseases and Colitis-Associated Colorectal Cancer[J]. Frontiers in Immunology, 2019,10.
[23] He Z, Chen L, Catalan-Dibene J, et al. Food colorants metabolizedj by commensal bacteria promote colitis in mice with dysregulated expression of interleukin-23[J]. Cell Metabolism, 2021,33(7):1358-1371.
[24] Saeedi B J, Kao D J, Kitzenberg D A, et al. HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity[J]. Mol Biol Cell, 2015,26(12):2252-2262.
[25] Xue X, Ramakrishnan S, Anderson E, et al. Endothelial PAS Domain Protein 1 Activates the Inflammatory Response in the Intestinal Epithelium to Promote Colitis in Mice[J]. Gastroenterology, 2013,145(4):831-841.
[26] W. K. Eddie Ip, Namiko Hoshi, Dror S. Shouval et al. Anti-inflammatory effect of IL-10 mediated by metabolic reprogramming of macrophages. Science, 05 May 2017, 356(6337):513-519, doi:10.1126/science.aal3535
[27] West N R, Hegazy A N, Owens B M J, et al. Oncostatin M drives intestinal inflammation and predicts response to tumor necrosis factor–neutralizing therapy in patients with inflammatory bowel disease[J]. Nature Medicine, 2017,23(5):579-589.
[28] Body-Malapel M, Djouina M, Waxin C, et al. The RAGE signaling pathway is involved in intestinal inflammation and represents a promising therapeutic target for Inflammatory Bowel Diseases[J]. Mucosal Immunol, 2019,12(2):468-478.
[29] Han W, Mercenier A, Ait Belgnaoui A, et al. Improvement of an experimental colitis in rats by lactic acid bacteria producing superoxide dismutase[J]. Inflammatory bowel diseases, 2006,12(11):1044-1052.
[30] Labbé A, Ganopolsky J G, Martoni C J, et al. Bacterial Bile Metabolising Gene Abundance in Crohn's, Ulcerative Colitis and Type 2 Diabetes Metagenomes[J]. PLoS ONE, 2014,9(12):e115175.
[31]郭士浩,陈善稳,潘义生,刘玉村,王鹏远.大肠杆菌Nissle 1917与肠屏障功能的研究进展[J].肠外与肠内营养,2018,25(03):184-187+192.
[33]Michael Schultz, MD, Dr habil (Germany), FRACP, Clinical use of E. coli Nissle 1917 in inflammatory bowel disease, Inflammatory Bowel Diseases, Volume 14, Issue 7, 1 July 2008, Pages 1012–1018
[34]Gosiewski, Tomasz; Strus, Magdalena; Fyderek, Krzysztof†; Kowalska-Duplaga, Kinga†; Wedrychowicz, Andrzej†; Jedynak-Wasowicz, Urszula‡; Sladek, Malgorzata†; Pieczarkowski, Stanislaw†; Adamski, Pawel§; Heczko, Piotr B.* Horizontal Distribution of the Fecal Microbiota in Adolescents With Inflammatory Bowel Disease, Journal of Pediatric Gastroenterology and Nutrition: January 2012 - Volume 54 - Issue 1 - p 20-27
[35]Weber, C. Lactococcus lactis alleviates oxidative stress and colitis in mice. Nat Rev Gastroenterol Hepatol 12, 429 (2015).
[36]Su Yong,Yao Wen,Huang Ruihua, et al.Effects of Sporolactobacillus S1 on the fluctuation of intestinal microflora and volatile fatty acid concentration of pre- and postweaning piglets. Journal of Fujian Agriculture and Forestry University. Natural Science Edition,2006,35(1):73-76. DOI:10.3969/j.issn.1671-5470.2006.01.017.
[37] Van Immerseel F, Ducatelle R, De Vos M, Boon N, Van De Wiele T, Verbeke K, Rutgeerts P, Sas B, Louis P, Flint HJ. Butyric acid-producing anaerobic bacteria as a novel probiotic treatment approach for inflammatory bowel disease. J Med Microbiol. 2010 Feb;59(Pt 2):141-143. doi: 10.1099/jmm.0.017541-0. Epub 2009 Nov 26. PMID: 19942690.
[38]Banasiewicz T, Domagalska D, Borycka-Kiciak K, Rydzewska G. Determination of butyric acid dosage based on clinical and experimental studies – a literature review. Gastroenterology Review/Przegląd Gastroenterologiczny. 2020;15(2):119-125. doi:10.5114/pg.2020.95556.
[39]Jing F, Cantu DC, Tvaruzkova J, Chipman JP, Nikolau BJ, Yandeau-Nelson MD, Reilly PJ. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity. BMC Biochem. 2011 Aug 10;12:44. doi: 10.1186/1471-2091-12-44. PMID: 21831316; PMCID: PMC3176148.
[40]Kallio, P., Pásztor, A., Thiel, K. et al. An engineered pathway for the biosynthesis of renewable propane. Nat Commun 5, 4731 (2014).
[41]. Shanahan F, Targan SR. Mechanisms of tissue injury in inflammatory bowel disease. In: Targan SR, Shanahan F, editors. Inflammatory Bowel Disease: From Bench to Bedside. Baltimore, Md: Williams & Wilkins; 1994. pp. 78–88.
[42]. Katz JA, Itoh J, Fiocchi C. Pathogenesis of inflammatory bowel disease. Current Opinion in Gastroenterology. 1999;15(4):291–297.
[43]. Podolsky DK. Inflammatory bowel disease (first of two parts) New England Journal of Medicine. 1991;325(13):928–937.
[44]. Isaacs KL, Sartor RB, Haskill S. Cytokine messenger RNA profiles in inflammatory bowel disease mucosa detected by polymerase chain reaction amplification. Gastroenterology. 1992;103(5):1587–1595.
[45]Szkaradkiewicz, Andrzej , et al. "Proinflammatory cytokines and IL-10 in inflammatory bowel disease and colorectal cancer patients." Archivum Immunologiae Et Therapiae Experimentalis 57.4(2009):291-294.
[46]Han, X., S. Ding, H. Jiang, and G. Liu, Roles of Macrophages in the Development and Treatment of Gut Inflammation. Frontiers in Cell and Developmental Biology, 2021. 9: p. 385.
[47]Chanput, W. , J. J. Mes , and H. J. Wichers . "THP-1 cell line: an in vitro cell model for immune modulation approach. " International Immunopharmacology 23.1(2014):37-45.
[48] Liu, X. , et al. "LPS‑induced proinflammatory cytokine expression in human airway epithelial cells and macrophages via NF‑κB, STAT3 or AP‑1 activation." Molecular Medicine Reports (2018).
[49]Mookherjee N, Brown KL, Bowdish DM, Doria S, Falsafi R, Hokamp K, Roche FM, Mu R, Doho GH, Pistolic J, Powers JP, Bryan J, Brinkman FS, Hancock RE. Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J Immunol. 2006 Feb 15;176(4):2455-64. doi: 10.4049/jimmunol.176.4.2455. PMID: 16456005.
[50]Koziel J, Bryzek D, Sroka A, Maresz K, Glowczyk I, Bielecka E, Kantyka T, Pyrć K, Svoboda P, Pohl J, Potempa J. Citrullination alters immunomodulatory function of LL-37 essential for prevention of endotoxin-induced sepsis. J Immunol. 2014 Jun 1;192(11):5363-72. doi: 10.4049/jimmunol.1303062. Epub 2014 Apr 25. PMID: 24771854; PMCID: PMC4036085.
[51]Pound LD, Patrick C, Eberhard CE, Mottawea W, Wang GS, Abujamel T, Vandenbeek R, Stintzi A, Scott FW. Cathelicidin Antimicrobial Peptide: A Novel Regulator of Islet Function, Islet Regeneration, and Selected Gut Bacteria. Diabetes. 2015 Dec;64(12):4135-47. doi: 10.2337/db15-0788. Epub 2015 Sep 14. PMID: 26370175.
[52]Kruidenier L, Kuiper I, Lamers CB, et al. Intestinal oxidative damage in inflammatory bowel disease: semi-quantification, localization, and association with mucosal antioxidants. J Pathol. 2003;201:28Y36.
[53]Seguí J, Gironella M, Sans M, Granell S, Gil F, Gimeno M, Coronel P, Piqué JM, Panés J. Superoxide dismutase ameliorates TNBS-induced colitis by reducing oxidative stress, adhesion molecule expression, and leukocyte recruitment into the inflamed intestine. J Leukoc Biol. 2004 Sep;76(3):537-44. doi: 10.1189/jlb.0304196. Epub 2004 Jun 14. PMID: 15197232.
[54]Han W, Mercenier A, Ait-Belgnaoui A, Pavan S, Lamine F, van Swam II, Kleerebezem M, Salvador-Cartier C, Hisbergues M, Bueno L, Theodorou V, Fioramonti J. Improvement of an experimental colitis in rats by lactic acid bacteria producing superoxide dismutase. Inflamm Bowel Dis. 2006 Nov;12(11):1044-52. doi: 10.1097/01.mib.0000235101.09231.9e. PMID: 17075345.
[55]Joyce, S. A., et al. (2014). "Bacterial bile salt hydrolase in host metabolism: Potential for influencing gastrointestinal microbe-host crosstalk." Gut Microbes 5(5): 669-674.
[56]Duboc H, Rajca S, Rainteau D, et al Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases Gut 2013;62:531-539.
[57]Nugent SG, Kumar D, Rampton DS, Evans DF. Intestinal luminal pH in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylates and other drugs. Gut. 2001 Apr;48(4):571-7. doi: 10.1136/gut.48.4.571. PMID: 11247905; PMCID: PMC1728243.
[58] Madsen S M, Arnau J, Vrang A, et al. Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of Lactococcus lactis. Molecular Microbiology, 1999, 32(1):75-87.
[59]Akyol, I., Comlekcioglu, U., Karakas, A. et al. Regulation of the acid induciblercfB promoter inLactococcus lactis subsp.lactis . Ann. Microbiol. 58, 269 (2008).
[60]Tian H, Tan J, Zhang L, et al. Increase of stress resistance in Lactococcus lactis via a novel food-grade vector expressing a shsp gene from Streptococcus thermophilus. Braz J Microbiol. 2012;43(3):1157-1164. doi:10.1590/S1517-838220120003000043
[61] Nigam A, Ziv T, Oron-Gottesman A, Engelberg-Kulka H2019. Stress-induced MazF-mediated proteins in Escherichia coli. mBio 10: e00340-19. doi:10.1128/mBio.00340-19.
[62] Govers MJ, Van der Meet R. Effects of dietary calcium and phosphate on the intestinal interactions between calcium, phosphate, fatty acids, and bile acids.Gut 1993;34:365-370.