Dual engineered bacteria improve inflammatory bowel disease in mice.

Currently, there are many different therapies available for inflammatory bowel disease (IBD), including engineered live bacterial therapeutics. However, most of these studies focus on producing a single therapeutic drug using individual bacteria, which may cause inefficacy. The use of dual drugs can enhance therapeutic effects. However, expressing multiple therapeutic drugs in one bacterial chassis increases the burden on the bacterium and hinders good secretion and expression. Therefore, a dual-bacterial, dual-drug expression system allows for the introduction of two probiotic chassis and enhances both therapeutic and probiotic effects. In this study, we constructed a dual bacterial system to simultaneously neutralize pro-inflammatory factors and enhance the anti-inflammatory pathway. These bacteria for therapy consist of Escherichia coli Nissle 1917 that expressed and secreted anti-TNF-α nanobody and IL-10, respectively. The oral administration of genetically engineered bacteria led to a decrease in inflammatory cell infiltration in colon and a reduction in the levels of pro-inflammatory cytokines. Additionally, the administration of engineered bacteria did not markedly aggravate gut fibrosis and had a moderating effect on intestinal microbes. This system proposes a dual-engineered bacterial drug combination treatment therapy for inflammatory bowel disease, which provides a new approach to intervene and treat IBD. KEY POINTS: • The paper discusses the effects of using dual engineered bacteria on IBD • Prospects of engineered bacteria in the clinical treatment of IBD.

Construction of an Engineered Escherichia coli with Efficient Chemotactic and Metabolizing Ability toward Tetrathionate.

The emergence of genetically engineered bacteria has provided a new means for the diagnosis and treatment of diseases. However, in vivo applications of these engineered bacteria are hindered by their inefficient accumulation in areas of inflammation. In this study, we constructed an engineered Escherichia coli (E. coli) for directional migration toward tetrathionate (a biomarker of gut inflammation), which is regulated by the TtrSR two-component system (TCS) from Shewanella baltica OS195 (S. baltica). Specifically, we removed endogenous cheZ to control the motility of E. coli. Moreover, we introduced the reductase gene cluster (ttrBCA) from Salmonella enterica serotype typhimurium (S. typhimurium), a major pathogen causing gut inflammation, into E. coli to metabolize tetrathionate. The resulting strain was tested for its motility along the gradients of tetrathionate; the engineered strain exhibits tropism to tetrathionate compared with the original strain. Furthermore, the engineered E. coli could only restore its smooth swimming ability when tetrathionate existed. With these modifications enabling tetrathionate-mediated chemotactic and metabolizing activity, this strategy with therapeutic elements will provide a great potential opportunity for target treatment of various diseases by swapping the corresponding genetic circuits.

Biological Sentinel: An Efficient Engineered Bacterial System for Monitoring and Tracing Regulated Hazardous Chemicals.

Monitoring and tracing of regulated hazardous chemicals is a public security issue of global concern. However, accurately recording historical exposure remains challenging. Here, we designed a Biological Sentinel System (BOSS) for in situ and long-term monitoring of hazardous chemical exposure using a chemical-induced base-editing system that activates antibiotic resistance screening, producing an obvious colorimetric signal. Exposure events can be written into an inheritable genomic DNA sequence, which can be read using gene sequencing. As a proof of concept, we demonstrated the accurate detection of cocaine and 2,4-dinitrotoluene using BOSS under simulated application scenarios. In addition, we integrated alternative biosensors to illustrate the modularity and extensibility of this monitoring platform. This work provides a promising paradigm for developing engineered microorganisms as an alternative to electronic monitors for regulated hazardous chemicals.

Protocol for engineering E. coli Nissle 1917 to diagnose, record, and ameliorate inflammatory bowel disease in mice.

Engineered microorganisms hold potential for disease diagnosis and treatment. Here, we present a protocol to engineer E. coli Nissle 1917 strain (iROBOT) using genome insertion and plasmid construction to diagnose, record, and ameliorate inflammatory bowel disease in mice. We describe steps for constructing and administering iROBOT, diagnosing and recording colitis, preparing samples, and analyzing fluorescence and base editing ratios of iROBOT. We detail a colitis ameliorating assay using the disease activity index, colon length, tissue pathological section, and cytokine analysis. For complete details of the use and execution of this protocol, please refer to Zou et al.1.

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.

Prebiotics-Controlled Disposable Engineered Bacteria for Intestinal Diseases.

As a new method of diagnosis and treatment for intestinal diseases, intelligent engineered bacteria based on synthetic biology have been developed vigorously in recent years. However, how to deal with the engineered bacteria in vivo after completing the tasks is an urgent problem to be resolved. In this study, we constructed a thiosulfate (a biomarker of inflammatory bowel disease)-responsive engineered bacteria to generate two signals, sfGFP (monitoring) and gain-of-function (translation activation) mutation (ACG to ATG), in the initiation codon of lysisE (recording) via the CRISPR/Cas9-mediated base editing system. Once these two signals were detected, xylose could be added to induce lysis E expression, resulting in the destruction of the edited bacteria and the release of AvCystain simultaneously. Overall, our innovative engineered bacteria can record instant and historical information of the disease, and especially, the edited bacteria can be artificially attenuated and release drug in situ when needed, ultimately serving as a disposable and recyclable candidate for more types of diseases.

Coupling split-lux cassette with a toggle switch in bacteria for ultrasensitive blood markers detection in feces and urine.

Cell-based biosensors have powerful abilities to sense a variety of signal chemical molecules. However, compared with commercialized methods, whole-cell biosensors cannot meet the requirements for the lower sensitivity and faster response. Here, we reprogrammed a gene circuit by coupling split-lux cassette with a toggle switch for detecting heme ultra-sensitively. The resultant biosensor (named YES601) exhibited improved detection limit (0.12 ppm) and satisfied maximum induction ratio (more than 4000 folds) for lysed blood detection. Furthermore, we harnessed YES601 to detect the blood signal in the human urine and feces from the mice with DSS-induced colitis, and the results indicated that YES601 showed more satisfied sensitivity and maximum induction ratio compared with chemical method. This ultrasensitive blood biosensor will be applied to detect trace blood in vitro for early-stage diagnosis of serious diseases, and aiding the rapid development for application in diagnosis in vivo in the future.

Design and optimization of E. coli artificial genetic circuits for detection of explosive composition 2,4-dinitrotoluene.

The detection of mine-based explosives poses a serious threat to the lives of deminers, and carcinogenic residues may cause severe environmental pollution. Whole-cell biosensors that can detect on-site in dangerous or inaccessible environments have great potential to replace conventional methods. Synthetic biology based on engineering modularity serves as a new tool that could be used to engineer microbes to acquire desired functions through artificial design and precise regulation. In this study, we designed artificial genetic circuits in Escherichia coli MG1655 by reconstructing the transcription factor YhaJ-based system to detect explosive composition 2,4-dinitrotoluene (2,4-DNT). These genetic circuits were optimized at the transcriptional, translational, and post-translational levels. The binding affinity of the transcription factor YhaJ with inducer 2,4-DNT metabolites was enhanced via directed evolution, and several activator binding sites were inserted in sensing yqjF promoter (PyqjF) to further improve the output level. The optimized biosensor PyqjF×2-TEV-(mYhaJ + GFP)-Ssr had a maximum induction ratio of 189 with green fluorescent signal output, and it could perceive at least 1 µg/mL 2,4-DNT. Its effective and robust performance was verified in different water samples. Our results demonstrate the use of synthetic biology tools to systematically optimize the performance of sensors for 2,4-DNT detection, that lay the foundation for practical applications.

Acylation driven by intracellular metabolites in host cells inhibits Cas9 activity used for genome editing.

CRISPR-Cas, the immune system of bacteria and archaea, has been widely harnessed for genome editing, including gene knockouts and knockins, single-base editing, gene activation, and silencing. However, the molecular mechanisms underlying fluctuations in the genome editing efficiency of crispr in various cells under different conditions remain poorly understood. In this work, we found that Cas9 can be ac(et)ylated by acetyl-phosphate or acyl-CoA metabolites both in vitro and in vivo. Several modifications are associated with the DNA or sgRNA binding sites. Notably, ac(et)ylation of Cas9 driven by these metabolites in host cells potently inhibited its binding and cleavage activity with the target DNA, thereby decreasing Crispr genome editing efficiency. This study provides more insights into understanding the effect of the intracellular environment on genome editing application of crispr with varying efficiency in hosts.

Long-Term Rewritable Report and Recording of Environmental Stimuli in Engineered Bacterial Populations.

DNA writing (living sensing recorders) based whole-cell biosensors can capture transient signals and then convert them into readable genomic DNA changes. The primitive signals can be easily obtained by sequencing technology or analysis of protein activity (such as fluorescent protein). However, the functions of the current living sensing recorders still need to be expanded, and the difficulty of rewriting in complex biological environments has further limited their applications. In this study, we designed a long-term rewritable recording system using a CRISPR base editor-based synthetic genetic circuit, named CRISPR-istop. This system can convert stimuli into changes in the fluorescence intensity (reporter) and single-base mutations in genomic DNA (recording). Furthermore, we updated the biological circuit through the strategy of coupling the single-base mutation (record site) and the loss-of-function of the targeted protein (translation stopped by stop codon introduction), and we can remove edited bacteria from a population through selective sweeps upon applying a selective pressure. It successfully conducted the rewritable reporter and recording of the nutrient arabinose and pollutant arsenite with two rounds of continuous operation (10 passages/round, 12 h/passage). These observations indicated that the CRISPR-istop system can report and record stimuli over time; moreover, the recording can be manually erased and rewritten as needed. This method has great potential to be extended to more complicated recording systems to execute sophisticated tasks in inaccessible environments for synthetic biology and biomedical applications, such as monitoring disease-relevant physiological markers or other molecules.