One-stop Solutions for Escherichia coli Nissle 1917
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Background of Escherichia coli Nissle 1917 (EcN)
Escherichia coli Nissle 1917 (EcN) is a well-known probiotic strain with multiple beneficial effects on intestinal homeostasis. It has been extensively characterized not only at the phenotypic level but also at the molecular genetic level. Engineered microorganisms are being rapidly developed for targeted therapy of disease. EcN is a genetically tractable probiotic with a well-established human safety record, it has recently emerged as a microbial chassis and a next-generation strategy for generating living therapeutics, drug delivery vehicles, and microbial platforms for industrial production and to modulate microbially derived metabolites in the gut.
Engineered microorganisms, such as the probiotic strain EcN, provide a strategy to detect and modulate concentrations of metabolites or therapeutic agents in the gastrointestinal tract. EcN has been used in several clinical trials for the treatment of gastrointestinal diseases. A plasmid-free EcN has been successfully constructed and termed EcNP. EcNP expressed more proteins than EcN, and the plasmid EcNP and pMT1 laid the foundation for the efficient engineering of E. coli Nissle as an active therapeutic drug. With discoveries about their biological functions, the genetic characteristics of EcN have been extensively studied.
Fig.1 Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology.1
One-stop Solutions for EcN Engineering
1. EcN Gene Knockout Services
1. clbp gene deletion mutant strain
ClbP is an inner-membrane D-aminopeptidase that cleaves precolibactin analogs with different acyl chains and amide substituts but is highly specific for the D-asparagine side chain. ClbP peptidase activity, which is essential for colibactin production, did not affect siderophore micromycin activity, providing a way to construct on-genotoxic strains with antimicrobial activity.
2. kpsM gene deletion mutant strain
kpsM is a member of the gene cluster responsible for capsular polysaccharide synthesis, which is an important virulence factor of bacteria and can destroy the host immune system. It has been shown that deletion of kpsM reduces the virulence of extraintestinal pathogenic Escherichia coli in pigs.
3. Genomic deletion on nlpI to obtain a hypervesiculation strain of EcN
Knockout of the nlpI gene has been reported to be safe for bacterial membrane integrity and stability.
4. curli deletion mutant strain
The probiotic EcN produces curli fibers on the bacterial surface, which are the major protein components of E. coli biofilms. The potential of curli-based pathogen isolation strategies could help develop novel enteric therapies based on VHH.
5. Construction of the minicell for delivery of self-generated azurin to cancer cell
Knockout of minCD and overexpression of minE in EcN can achieve large-scale production of minicells. The use of this simple, low-cost strategy to transport proteins holds promise for a variety of therapeutic applications.
6. Elimination of endogenous plasmids (Plasmid-free strains)
Engineered probiotic EcN is expected to be used in the diagnosis and treatment of a variety of diseases. In EcN, two stably replicating cryptic plasmids, pMUT1 and pMUT2, were identified, creating a metabolic load that limits their use in genetic engineering. However, introduced plasmids often require antibiotics to maintain genetic stability, and hidden plasmids in EcN are usually removed to avoid plasmid incompatibility altering intrinsic probiotic properties. Endogenous EcN plasmids can be genetically manipulated and stably maintained.
7. Other customized projects according to the client's requirements.
2. Gene Knock-in/Recombinant Strain Construction Services
1. Construction of a sustainable 3-hydroxybutyrate-producing probiotic Escherichia coli
(R) -β-hydroxybutyric acid (3HB) is the main component of ketone bodies in animals, which can be used as an energy source during starvation or exercise, and is also considered as a therapeutic agent. Beneficial effects of 3HB in the treatment of neurodegenerative diseases or hypertension, seizures, and NLRP3-mediated inflammation have been reported. Recently, 3HB was shown to ameliorate dextran sulfate sodium (DSS) -induced colitis in animal models, and since EcN is commonly used in the treatment of inflammatory bowel disease (IBD), the combination of EcN with therapeutic compounds and probiotics may show synergistic effects to enhance the therapeutic effects of colitis.
2. EcN-derived TFF-fused curli construction
The programmed bacteria assemble a multivalent material in the gut and are decorated with anti-inflammatory domains, and the domains shown are designed to target the material to the mucosal layer of the epithelium and promote host processes that enhance epithelial barrier function. This scaffold material produced by bacteria is based on coiled fibers, which are common protein components in the bacterial extracellular matrix. It has been shown that therapeutic protein matrices can be produced in situ using beneficial bacteria.
3. ECN-pE construction
By studying probiotics overexpressing catalase and superoxide dismutase (ECN-pe), it was found that in a mouse IBD model induced by different chemical drugs, chitosan/sodium alginate coating ECN-pE (ECN-pE(C/A)2) effectively relieved inflammation and repaired epithelial barriers in the colon.
4. For gamma-aminobutyric acid (GABA) production
GABA has been shown to affect neurological disorders, including mood and sleep disorders as well as epilepsy, depression, and anxiety. It is a non-essential amino acid produced by bacteria in the human gastrointestinal tract and acts as a neurotransmitter,
5. Recombinant production of omega-3 fatty acids
Omega-3 fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are beneficial to human health. EcN for EPA/DHA production or fat isolated from gEcN may be an economical, sustainable, and convenient source for the industrial production of omega-3 fatty acids for human consumption and pharmaceutical applications.
6. Engineering EcN against P. aeruginosa infection
By generating a synthetic genetic system including a gene encoding an antibiofilm enzyme and using the probiotic strain EcN as the host.
7. Engineering EcN against acute colitis
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Elafin-expressing EcN protects against DSS-induced acute colitis in mice and may be an effective and cost-effective treatment for IBD.
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Construction of EcN strain (iROBOT) to ameliorate IBD.
8. Construct a tumor-targeting engineered probiotic
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Azurin has been proposed as a promising tumoricidal protein. HlpA is an unanchored colorectal cancer cell surface protein of S. gallolyticus with a submicromolar affinity for colorectal cancer cell lines. An engineered probiotic Ep-AH expressing both HlpA and Azurin based on the "targeted killing" strategy can be constructed.
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Expression of therapeutic proteins in EcN, e.g. IL-2.
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Expression of prodrug-converting enzymes in EcN
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Engineering of EcN-derived minicells
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Engineering of EcN bacterial ghosts
9. Construct EcN-GLP-1 strain against gut-brain disorders
Glucagon-like peptide-1 (GLP-1), the prototype of intestinal blood glucose physiologically released by enteroendocrine L cells, is essential for glucose homeostasis and is used to treat diabetes in response to food intake and metabolic regulation.
10. Other customized projects according to the client's requirements.
3. Engineered EcN Bacteria as Imaging Vectors
Recent advances in biotechnology have enabled bacteria-enhanced medical imaging. The success of bacteria-enhanced medical imaging depends on the accumulation of local bacteria at the lesion site, and EcN is often used for gastrointestinal (GI) applications due to its ability to colonize the GI tract.
Technology
Using CRISPR technology, the fluorescent protein expression cassette was integrated into the genome of probiotic Escherichia coli Nissle1917 to construct a fluorescent-labeled strain.
Optical Imaging
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Fluorescence imaging (FI)
The combination of selective aggregation of bacteria at disease sites and genetic engineering, combined with fluorescent protein reporter genes, provides a targeted, noninvasive tool for detection and monitoring. Due to the inherent cancer-targeting ability of bacteria, many research efforts have focused on the use of FI to detect bacteria for cancer and therapeutic effect monitoring.
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Bioluminescence
Bioluminescence imaging is achieved by genetically engineering bacteria to contain bioluminescence reporter genes such as luciferase, which enables the visualization of bacteria.
Fluorescent Proteins
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sfGFP and YFP are suitable for imaging bacteria alone, or bacterial interacting cells or superficial tissues in animal models of bacterial infection.
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iRFP is suitable for bacterial disturbance animal models and in vivo imaging of deep tissues and organs.
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Luc luciferase is the best choice for cell animal in vivo imaging observation.
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Lux bacterial luciferase, which is particularly suitable for long-term in vivo imaging observations in animal models of bacteria infection.
Escherichia coli Nissle1917 Specific Fluorescent Expression Vectors
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pBbE8a-RFP vector
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pM1s3AsG vector
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pM1s3TsR vector
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pM2s2TsR vector
Highlights
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Record-proven expertise and experience in probiotics
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Excellent efficiency and fast turnaround probiotic vector construction and gene editing
Creative Biolabs has a professional technical platform in the construction of probiotic engineering strains and has provided clients with gene knockout and insertion services for EcN strains. Our services are tailored to the specific needs of each client.
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LBSX-0522-GF116
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Escherichia coli Nissle 1917
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Escherichia coli Nissle 1917 is a probiotic strain with proven efficacy in inducing and maintaining remission of ulcerative colitis.
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Escherichia coli Nissle 1917 △pMUT1△pMUT2
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It is subjected to gene modification to remove two intracellular cryptic plasmids pMUT1 and pMUT2.
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Case Studies
Case 1: Construction of a Sustainable 3-hydroxybutyrate-producing Probiotic Escherichia coli Nissle 1917
Summary
Verified by PCR and sequencing, the strains E. coli Nissle 1917△l**A and E. coli Nissle 1917△l**A malEK: t**B-p**A-p**B have been correctly constructed.
Methods
Gene editing Method
Results
Fig.2 Identification of gene knock-out.
Fig.3 Identification of genes knock-in.
Deliverables
The delivered strains are freshly activated glycerol bacteria.
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E. coli Nissle 1917 (Original strain).
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E. coli Nissle 1917 knock-out strain.
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E. coli Nissle 1917 knock-in strain.
Brochures
Frequently Asked Questions
What are the Characteristics of Escherichia coli Strain Nissle 1917?
It can colonize the intestinal tract, has antagonistic activity, and anti-invasion ability, can synthesize endogenous antimicrobial peptides, promote mucosal integrity, enhance epithelial barrier function, anti-inflammatory and immunomodulatory effects, and stimulate the colonic mucosa.
What are the Potential New Ways to Use the Escherichia coli Nissle 1917 strain?
This strain is currently being investigated not only for its potential probiotic use but also because it may be a way to better deliver drugs to IBD. Researchers engineered a strain of E. coli (Nissel 1917) to secrete proteins of therapeutic value, which could help deliver drugs better by creating a new drug-delivery system. This modified E. coli delivery method has the power to improve outcomes in patients taking anti-TNF drugs.
Creative Biolabs is positioned to be a global leader in preclinical research in the field of live Biotherapeutics or probiotics. We have extensive project experience in the construction of engineered strains of Escherichia coli Nissle1917. Please feel free to contact us for more details.
Reference
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Ba, Fang, et al. "Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology." Biotechnology Journal 19.1 (2024): 2300327.
For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.
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