Engineered probiotics.

Engineered probiotics are a kind of new microorganisms produced by modifying original probiotics through gene editing. With the continuous development of tools and technology progresses, engineering renovation of probiotics are becoming more diverse and more feasible. In the past few years there have been some advances in the development of engineered probiotics that will benefit humankind. This review briefly introduces the theoretical basis of gene editing technology and focuses on some recent engineered probiotics researches, including inflammatory bowel disease, bacterial infection, tumor and metabolic diseases. It is hoped that it can provide help for the further development of genetically modified microorganisms, stimulate the potential of engineered probiotics to treat intractable diseases, and provide new ideas for the diagnosis of some diseases or some industrial production.

Empowering probiotics with high xanthine transport for effective hyperuricemia management.

Hyperuricemia, a prevalent metabolic disorder, poses a susceptibility to various complications. The conventional pharmacotherapeutic approaches for hyperuricemia often entail notable adverse effects, posing substantial clinical challenges. Hence, the imperative lies in the development of novel, safe and effective strategies for preventing and treating hyperuricemia. Here, we developed a probiotic Escherichia coli Nissle 1917 strain, designated as YES301, which contains a rationally designed xanthine importer XanQ, enabling efficient uptake of xanthine and hypoxanthine, consequently leading to reduced serum uric acid concentrations and amelioration of renal impairments in a murine model of hyperuricemia. Importantly, YES301 exhibited a therapeutic efficacy comparable to allopurinol, a conventional uric acid-lowering agent, and manifesting fewer adverse effects and enhanced biosafety. These findings highlight the promising potential of engineered probiotics in the management of hyperuricemia through reducing intestinal purine levels.

Spatiotemporal Delivery of Dual Nanobodies by Engineered Probiotics to Reverse Tumor Immunosuppression via Targeting Tumor-Derived Exosomes.

The anti-PD-L1 and its bispecific antibodies have exhibited durable antitumor immunity but still elicit immunosuppression mainly caused by tumor-derived exosomes (TDEs), leading to difficulty in clinical transformation. Herein, engineered Escherichia coli Nissle 1917 (EcN) coexpressing anti-PD-L1 and anti-CD9 nanobodies (EcN-Nb) are developed and decorated with zinc-based metal-organic frameworks (MOFs) loaded with indocyanine green (ICG), to generate EcN-Nb-ZIF-8CHO-ICG (ENZC) for spatiotemporal lysis of bacteria for immunotherapy. The tumor-homing hybrid system can specifically release nanobodies in response to near-infrared (NIR) radiation, thereby targeting TDEs and changing their biological distribution, remodeling tumor-associated macrophages to M1 states, activating more effective and cytotoxic T lymphocytes, and finally, leading to the inhibition of tumor proliferation and metastasis. Altogether, the microfluidic-enabled MOF-modified engineered probiotics target TDEs and activate the antitumor immune response in a spatiotemporally manipulated manner, offering promising TDE-targeted immune therapy.

Engineered probiotic cocktail with two cascade metabolic Escherichia coli for the treatment of hyperlysinemia.

Engineering probiotics have emerged as a potential strategy for the treatment of metabolic diseases. However, due to the exceptional complexity of these metabolic disorders and the intricate relationship between gut microbes, it is difficult to achieve an ideal therapeutic effect in a specific metabolic disorder using only a single engineered strain. In this work, we proposed a probiotic cocktail strategy by engineering two cascade metabolic bacteria to treat hyperlysinemia, an inherited lysine metabolic disorder with loss of α-aminoadipate semialdehyde synthase (AASS) activity. A probiotic E. coli Nissle 1917 strain EcNT (pTLS) with a heterologous enzyme pathway in Saccharomyces cerevisiae was engineered to metabolize the excess lysine. Another one EcNT (pK25) was engineered to consume the products of lysine metabolism. The bacterial cocktail enables the maintenance of a metabolic cascade with AASS-like functional activity to maintain the blood lysine concentrations and downstream metabolites. In vitro experimental results showed that the cocktail bacteria had a better metabolic capacity and metabolites balance at a ratio of EcNT (pTLS) and EcNT (pK25) of 1:2. Feeding of the cocktail bacteria to the mouse model effectively reduced the concentration of lysine and balanced saccharopine in the plasma of hyperlysinemia-like mice. These findings not only provide a promising strategy of probiotic stains for the treatment of hyperlysinemia but also highlight the potential of engineered cascade cocktails to intervene and even cure other inherited metabolic diseases.

A synthetic microbial consortium protects against obesity by regulating vitamin B6 metabolism.

Constructing synthetic microbial consortia is a challenging task but holds enormous potential for various applications. Our previous droplet-based microfluidic approach allowed for the isolation of bacteria that could utilize metabolites from an engineered bacterium BsS-RS06551 with anti-obesity potential, facilitating the construction of synthetic microbial consortia. Here, we identified a strain of Bifidobacterium pseudocatenulatum JJ3 that interacted with BsS-RS06551, and in vitro coculture showed that BsS-RS06551 was likely to interact with JJ3 through five dipeptides. Pathway analysis revealed that the vitamin B6 metabolism pathway was enriched in the coculture of BsS-RS06551 and JJ3 compared with the individual culture of BsS-RS06551. Additionally, we confirmed that the administration of JJ3 significantly alleviated obesity and related disorders in mice fed a high-fat diet. Notably, continuous ingestion of the synthetic microbial consortium comprising BsS-RS06551 and JJ3 not only exhibited a more pronounced impact on alleviating obesity compared to the individual administration of BsS-RS06551 or JJ3 but also enriched the population of Bifidobacterium longum and perturbed the vitamin B6 metabolism pathway in the gut. Synthetic microbial consortia represent a promising frontier for synthetic biology, and our strategy provides guidance for constructing and applying such consortia.

Engineered probiotics with sustained release of interleukin-2 for the treatment of inflammatory bowel disease after oral delivery.

Inflammatory bowel disease (IBD) is a kind of auto-immune disease characterized by disrupted intestinal barrier and mucosal epithelium, imbalanced gut microbiome and deregulated immune responses. Therefore, the restoration of immune equilibrium and gut microbiota could potentially serve as a hopeful approach for treating IBD. Herein, the oral probiotic Escherichia coli Nissle 1917 (ECN) was genetically engineered to express secretable interleukin-2 (IL-2), a kind of immunomodulatory agent, for the treatment of IBD. In our design, probiotic itself has the ability to regulate the gut microenvironment and IL-2 at low dose could selectively promote the generation of regulatory T cells to elicit tolerogenic immune responses. To improve the bioavailability of ECN expressing IL-2 (ECN-IL2) in the gastrointestinal tract, enteric coating Eudragit L100-55 was used to coat ECN-IL2, achieving significantly enhanced accumulation of engineered probiotics in the intestine. More importantly, L100-55 coated ECN-IL2 could effectively activated Treg cells to regulate innate immune responses and gut microbiota, thereby relieve inflammation and repair the colon epithelial barrier in dextran sodium sulfate (DSS) induced IBD. Therefore, genetically and chemically modified probiotics with excellent biocompatibility and efficiency in regulating intestinal microflora and intestinal inflammation show great potential for IBD treatment in the future.

Biochemotaxis-Oriented Engineering Bacteria Expressing GLP-1 Enhance Diabetes Therapy by Regulating the Balance of Immune.

Glucagon like peptide-1 (GLP-1) is an effective hypoglycemic drug that can repair the pancreas ß cells and promote insulin secretion. However, GLP-1 has poor stability and lacks of target ability, which makes it difficult to reach the site of action to exert its efficacy. Here, GLP-1-expressing plasmids are introduced into the Escherichia coli Nissle 1917 (EcN) and a lipid membrane is formed through simple self-assembly on its surface, resulting in an oral delivery system (LEG) capable of resisting the harsh environment of the gastrointestinal tract. The system utilizes the chemotactic properties of probiotics to achieve efficient enrichment at the pancreatic site, and protects islet ß cells from destruction by regulating the balance of immune cells. More interestingly, LEG not only continuously produces GLP-1 to restore pancreatic islet ß cell function and secrete insulin to control blood sugar levels, but also regulates the intestinal flora and increases the richness and diversity of probiotics. In mice diabetes models, oral administration of LEG only once every other day has good biosafety and compliance, and achieves long-term control of blood glucose. Therefore, this strategy not only provides an oral delivery platform for pancreatic targeting, but also opens up new avenues for reversing diabetes.

T cell cascade regulation initiates systemic antitumor immunity through living drug factory of anti-PD-1/IL-12 engineered probiotics.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but only works in a subset of patients due to the insufficient infiltration, persistent exhaustion, and inactivation of T cells within a tumor. Herein, we develop an engineered probiotic (interleukin [IL]-12 nanoparticle Escherichia coli Nissle 1917 [INP-EcN]) acting as a living drug factory to biosynthesize anti-PD-1 and release IL-12 for initiating systemic antitumor immunity through T cell cascade regulation. Mechanistically, INP-EcN not only continuously biosynthesizes anti-PD-1 for relieving immunosuppression but also effectively cascade promote T cell activation, proliferation, and infiltration via responsive release of IL-12, thus reaching a sufficient activation threshold to ICB. Tumor targeting and colonization of INP-EcNs dramatically increase local drug accumulations, significantly inhibiting tumor growth and metastasis compared to commercial inhibitors. Furthermore, immune profiling reveals that anti-PD-1/IL-12 efficiently cascade promote antitumor effects in a CD8+ T cell-dependent manner, clarifying the immune interaction of ICB and cytokine activation. Ultimately, such engineered probiotics achieve a potential paradigm shift from T cell exhaustion to activation and show considerable promise for antitumor bio-immunotherapy.

Intranasal administration of Escherichia coli Nissle expressing the spike protein of SARS-CoV-2 induces long-term immunization and prevents spike protein-mediated lung injury in mice.

While current anti-Spike protein (SP) vaccines have been pivotal in managing the pandemic, their limitations in delivery, storage, and the inability to provide mucosal immunization (preventing infections) highlight the ongoing necessity for research and innovation. To tackle these constraints, our research group developed a bacterial-based vaccine using a non-pathogenic E. coli Nissle 1917 (EcN) strain genetically modified to express the SARS-CoV-2 spike protein on its surface (EcN-pAIDA1-SP). We intranasally delivered the EcN-pAIDA1-SP in two doses and checked specific IgG/IgA production as well as the key immune mediators involved in the process. Moreover, following the initial and booster vaccine doses, we exposed both immunized and non-immunized mice to intranasal delivery of SARS-CoV-2 SP to assess the effectiveness of EcN-pAIDA1-SP in protecting lung tissue from the inflammation damage. We observed detectable levels of anti-SARS-CoV-2 spike IgG in serum samples and IgA in bronchoalveolar lavage fluid two weeks after the initial treatment, with peak concentrations in the respective samples on the 35th day. Moreover, immunoglobulins displayed a progressively enhanced avidity index, suggesting a selective binding to the spike protein. Finally, the pre-immunized group displayed a decrease in proinflammatory markers (TLR4, NLRP3, ILs) following SP challenge, compared to the non-immunized groups, along with better preservation of tissue morphology. Our probiotic-based technology provides an effective immunobiotic tool to protect individuals against disease and control infection spread.

Surface Display of Duck Hepatitis A Virus Type 1 VP1 Protein on Bacillus subtilis Spores Elicits Specific Systemic and Mucosal Immune Responses on Mice.

Duck viral hepatitis, primarily caused by duck hepatitis A virus type 1 (DHAV-1), poses a significant threat to the global duck industry. Bacillus subtilis is commonly utilized as a safe probiotic in the development of mucosal vaccines. In this study, a recombinant strain of B. subtilis, designated as B. subtilis RV, was constructed to display the DHAV-1 capsid protein VP1 on its spore surface using the outer coat protein B as an anchoring agent. The immunogenicity of this recombinant strain was evaluated in a mouse model through mixed feeding immunization. The results indicated that B. subtilis RV could elicit specific systemic and mucosal immune responses in mice, as evidenced by the high levels of serum IgG, intestinal secretory IgA, and potent virus-neutralizing antibodies produced. Furthermore, the recombinant strain significantly upregulated the expression levels of IL-2, IL-6, IL-10, TNF-α, and IFN-γ in the intestinal mucosa. Thus, the recombinant strain maintained the balance of the Th1/Th2 immune response and demonstrated an excellent mucosal immune adjuvant function. In summary, this study suggests that B. subtilis RV can be a novel alternative for effectively controlling DHAV-1 infection as a vaccine-based feed additive.

A red light-controlled probiotic bio-system for in-situ gut-brain axis regulation.

Microbes regulate brain function through the gut-brain axis, deriving the technology to modulate the gut-brain axis in situ by engineered probiotics. Optogenetics offers precise and flexible strategies for controlling the functions of probiotics in situ. However, the poor penetration of most frequently used short wavelength light has limited the application of optogenetic probiotics in the gut. Herein, a red-light optogenetic gut probiotic was applied for drug production and delivery and regulation of the host behaviors. Firstly, a Red-light Optogenetic E. coli Nissle 1917 strain (ROEN) that could respond to red light and release drug product by light-controlled lysis was constructed. The remaining optical power of red light after 3 cm tissue was still able to initiate gene expression of ROEN and produce about approximately 3-fold induction efficiency. To give full play to the in vivo potential of ROEN, its responsive ability of the penetrated red light was tested, and its encapsulation was realized by PH-sensitive alginate microcapsules for further oral administration. The function of ROEN for gut-brain regulation was realized by releasing Exendin-4 fused with anti-neonatal Fc receptor affibody. Neuroprotection and behavioral regulation effects were evaluated in the Parkinson's disease mouse model, after orally administration of ROEN delivering Exendin-4 under optogenetic control in the murine gut. The red-light optogenetic probiotic might be a perspective platform for in situ drug delivery and gut-brain axis regulation.

Biomarker-responsive engineered probiotic diagnoses, records, and ameliorates inflammatory bowel disease in mice.

Rapid advances in synthetic biology have fueled interest in engineered microorganisms that can diagnose and treat disease. However, designing bacteria that detect dynamic disease-associated biomarkers that then drive treatment remains difficult. Here, we have developed an engineered probiotic that noninvasively monitors and records inflammatory bowel disease (IBD) occurrence and progression in real time and can release treatments via a self-tunable mechanism in response to these biomarkers. These intelligent responsive bacteria for diagnosis and therapy (i-ROBOT) consists of E. coli Nissle 1917 that responds to levels of the inflammatory marker thiosulfate by activating a base-editing system to generate a heritable genomic DNA sequence as well as producing a colorimetric signal. Fluctuations in thiosulfate also drive the tunable release of the immunomodulator AvCystatin. Orally administering i-ROBOT to mice with colitis generated molecular recording signals in processed fecal and colon samples and effectively ameliorated disease. i-ROBOT provides a promising paradigm for gastrointestinal and other metabolic disorders.

Oral Immunization with Escherichia coli Nissle 1917 Expressing SARS-CoV-2 Spike Protein Induces Mucosal and Systemic Antibody Responses in Mice.

As of October 2022, the COVID-19 pandemic continues to pose a major public health conundrum, with increased rates of symptomatic infections in vaccinated individuals. An ideal vaccine candidate for the prevention of outbreaks should be rapidly scalable, easy to administer, and able to elicit a potent mucosal immunity. Towards this aim, we proposed an engineered Escherichia coli (E. coli) Nissle 1917 (EcN) strain with SARS-CoV-2 spike protein (SP)-coding plasmid, which was able to expose SP on its cellular surface by a hybridization with the adhesin involved in diffuse adherence 1 (AIDA1). In this study, we presented the effectiveness of a 16-week intragastrically administered, engineered EcN in producing specific systemic and mucosal immunoglobulins against SARS-CoV-2 SP in mice. We observed a time-dependent increase in anti-SARS-CoV-2 SP IgG antibodies in the sera at week 4, with a titre that more than doubled by week 12 and a stable circulating titre by week 16 (+309% and +325% vs. control; both p < 0.001). A parallel rise in mucosal IgA antibody titre in stools, measured via intestinal and bronchoalveolar lavage fluids of the treated mice, reached a plateau by week 12 and until the end of the immunization protocol (+300, +47, and +150%, at week 16; all p < 0.001 vs. controls). If confirmed in animal models of infection, our data indicated that the engineered EcN may be a potential candidate as an oral vaccine against COVID-19. It is safe, inexpensive, and, most importantly, able to stimulate the production of both systemic and mucosal anti-SARS-CoV-2 spike-protein antibodies.

Programming therapeutic probiotics by self-tunable sense-and-respond genetic circuits.

Probiotics can be programmed to sense and respond to intracellular disease signals to deliver the desired therapeutic effectors. The sense-and-respond genetic circuits, especially self-tunable ones, hold promise in improving the precision, effectiveness, and intelligence of therapeutic activities. Here, we present notable advances in the creation of engineered probiotics that harbour sense-and-respond genetic circuits.

Novel Genetically Engineered Probiotics for Targeted Elimination of Pseudomonas aeruginosa in Intestinal Colonization.

The intestinal carriage rates of Pseudomonas aeruginosa are notably elevated in immunosuppressed individuals and hospitalized patients, increasing the risk of infection and antibiotic-associated diarrhea. A potential solution to this issue lies in autonomous antibacterial therapy, remaining inactive until a pathogen is detected, and releasing antibacterial compounds on demand to eliminate the pathogen. This study focuses on the development of genetically engineered probiotics capable of detecting and eradicating P. aeruginosa by producing and secreting PA2-GNU7, a P. aeruginosa-selective antimicrobial peptide (AMP), triggered by the presence of P. aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (3OC12HSL). To achieve this goal, plasmid-based systems were constructed to produce AMPs in response to 3OC12HSL and secrete them into the extracellular medium using either the microcin V secretion system or YebF as a carrier protein. Following the transfer of these plasmid-based systems to Escherichia coli Nissle 1917 (EcN), we successfully demonstrated the ability of the engineered EcN to express and secrete PA2-GNU7, leading to the inhibition of P. aeruginosa growth in vitro. In addition, in a mouse model of intestinal P. aeruginosa colonization, the administration of engineered EcN resulted in reduced levels of P. aeruginosa in both the feces and the colon. These findings suggest that engineered EcN holds promise as a potential option for combating intestinal P. aeruginosa colonization, thus mitigating the risk of future endogenous infections in vulnerable patients.

Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet. / 工程枯草芽孢杆菌通过Nrf2-Keap1途径缓解ETEC K88æ„ŸæŸ“ä»”çŒªå¯¼è‡´çš„è‚ é“æ°§åŒ–æŸä¼¤.

Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties. In this study, we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32 (WB800-KR32) using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2 (Nrf2)|-Kelch-like ECH-associated protein 1 (Keap1) pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli (ETEC) K88 in weaned piglets. Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet. The feed of the control group (CON) was infused with normal sterilized saline; meanwhile, the ETEC, ETEC+WB800, and ETEC+WB800-KR32 groups were orally administered normal sterilized saline, 5×1010 CFU (CFU: colony forming units) WB800, and 5×1010 CFU WB800-KR32, respectively, on Days 1|‒|14 and all infused with ETEC K88 1×1010 CFU on Days 15|‒|17. The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance, improved the mucosal activity of antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) and decreased the content of malondialdehyde (MDA). More importantly, WB800-KR32 downregulated genes involved in antioxidant defense (GPx and SOD1). Interestingly, WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum. WB800-KR32 markedly changed the richness estimators (Ace and Chao) of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces. The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway, providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.

Next-generation probiotics as a therapeutic strategy for the treatment of phenylketonuria: a review.

Phenylketonuria (PKU) is a rare genetic disease that causes brain toxicity due to the inability of the body to convert dietary phenylalanine to tyrosine by the action of phenylalanine hydroxylase. The only treatment for PKU so far is lifelong dietary intervention to ensure normal human growth and neurodevelopment. However, in adults, low long-term adherence to this type of dietary intervention has been observed. Given the important role of the intestinal microbiota in the process of digestion and disease prevention, probiotics could be a therapeutic strategy to help degrade dietary phenylalanine, reducing its levels before ingestion. Genetically modified probiotics designed as live biotherapeutic agents for the treatment of specific diseases are sophisticated alternative therapeutic strategies. In this review, the focus is on demonstrating what has been elucidated so far about the use of next-generation probiotics as a therapeutic strategy in the treatment of individuals with PKU. The results described in the literature are encouraging and use genetically modified engineered probiotics showing efficacy both in vitro and in vivo. These probiotics appear to be suitable for meeting the unmet need for new drugs for PKU.

Intestinal Engineered Probiotics as Living Therapeutics: Chassis Selection, Colonization Enhancement, Gene Circuit Design, and Biocontainment.

Intestinal probiotics are often used for the in situ treatment of diseases, such as metabolic disorders, tumors, and chronic inflammatory infections. Recently, there has been an increased emphasis on intelligent, customized treatments with a focus on long-term efficacy; however, traditional probiotic therapy has not kept up with this trend. The use of synthetic biology to construct gut-engineered probiotics as live therapeutics is a promising avenue in the treatment of specific diseases, such as phenylketonuria and inflammatory bowel disease. These studies generally involve a series of fundamental design issues: choosing an engineered chassis, improving the colonization ability of engineered probiotics, designing functional gene circuits, and ensuring the safety of engineered probiotics. In this review, we summarize the relevant past research, the progress of current research, and discuss the key issues that restrict the widespread application of intestinal engineered probiotic living therapeutics.

Hypoxic Tumor Radiosensitization Using Engineered Probiotics.

Owing to their ability to rapidly proliferate in specific niches and their amenability to genetic manipulation, bacteria are frequently studied as potential diagnostic or therapeutic bioagents in a range of pathological contexts. A sustained oxygen supply within solid tumors is essential in order to achieve positive radiotherapy (RT) outcomes, as these intratumoral oxygen levels are necessary to facilitate RT-induced reactive oxygen species (ROS) generation. In this study, a genetically engineered variant of the tumor-targeting probiotic E. coli Nissle 1917 bacteria that secret catalase is utilized to alleviate intratumoral hypoxia and to thereby enhance tumor radiosensitivity. These engineered bacteria are able to facilitate robust O2 evolution and consequent ROS generation in response to X-ray irradiation both in vitro and in vivo, significantly inhibiting tumor growth. Overall, the study highlights a novel and practical approach to enhance the efficacy of tumor RT, underscoring the value of future research in the field of probiotic medicine.

In vitro fermentation test bed for evaluation of engineered probiotics in polymicrobial communities.

In vitro fermentation systems offer significant opportunity for deconvoluting complex metabolic dynamics within polymicrobial communities, particularly those associated with the human gut microbiome. In vitro gut models have broad experimental capacity allowing rapid evaluation of multiple parameters, generating knowledge to inform design of subsequent in vivo studies. Here, our method describes an in vitro fermentation test bed to provide a physiologically-relevant assessment of engineered probiotics circuit design functions. Typically, engineered probiotics are evaluated under pristine, mono- or co-culture conditions and transitioned directly into animal or human studies, commonly resulting in a loss of desired function when introduced to complex gut communities. Our method encompasses a systematic workflow entailing fermentation, molecular and functional characterization, and statistical analyses to validate an engineered probiotic's persistence, plasmid stability and reporter response. To demonstrate the workflow, simplified polymicrobial communities of human gut microbial commensals were utilized to investigate the probiotic Escherichia coli Nissle 1917 engineered to produce a fluorescent reporter protein. Commensals were assembled with increasing complexity to produce a mock community based on nutrient utilization. The method assesses engineered probiotic persistence in a competitive growth environment, reporter production and function, effect of engineering on organism growth and influence on commensal composition. The in vitro test bed represents a new element within the Design-Build-Test-Learn paradigm, providing physiologically-relevant feedback for circuit re-design and experimental validation for transition of engineered probiotics to higher fidelity animal or human studies.