CD Biosynsis offers high-efficiency Bacillus subtilis Gene Knockout Services, a foundational tool for microbial engineering, metabolic pathway optimization, and functional genomics studies. B. subtilis, being a Gram-positive bacterium, utilizes natural competence for DNA uptake, which is leveraged for targeted genomic modification via homologous recombination. Our service provides precise and stable deletion of single or multiple target genes, crucial for eliminating unwanted proteases, diverting metabolic flux towards target products, or studying gene function. We employ robust techniques, including plasmid-based gene replacement and marker-less deletion strategies, ensuring the delivery of genetically stable, fully validated knockout strains for research and industrial applications.
Get a QuoteGene knockout in B. subtilis is highly effective due to its inherent ability to readily incorporate exogenous DNA, making it a powerful platform for engineering. Our methodologies focus on creating precise, irreversible deletions, typically replacing the target gene with a selection marker, which is then often cured to create a clean, marker-less strain suitable for iterative Multi-Gene Knockout. Whether the goal is to enhance the production of secreted proteins by eliminating native proteases (e.g., AprE, NprE) or to boost the yield of a specific metabolite by disrupting a competing pathway, our tailored approach ensures the highest rate of success and genetic stability, providing reliable foundation strains for further Pathway Optimization.
Deletion Target Identification
Consultation to select single or multiple genes (e.g., protease, metabolic shunt) for deletion to meet project goals.
Homology Arm Design
Synthesis of DNA fragments with appropriate homology arms (typically 500-1500 bp) flanking the target gene for efficient recombination.
Selection Marker Selection
Choice of selection markers (e.g., antibiotic resistance) compatible with the B. subtilis host and the subsequent marker-curing strategy.
Competence Induction
Optimization of growth conditions to maximize the natural competence of B. subtilis cells for efficient transformation with the deletion cassette.
Targeted Deletion and Selection
Transformation of the deletion cassette followed by stringent antibiotic selection to isolate colonies where homologous recombination successfully occurred.
Single vs. Multi-Gene Knockout
Strategy development for sequential deletion of multiple genes, ensuring the selection markers are compatible or reusable (see Multi-Gene Knockout Strain Construction).
Selection Marker Removal
Use of counter-selection (e.g., use of sucrose sensitivity genes) or site-specific recombinases (Cre/Lox) to remove the antibiotic marker cleanly.
PCR Verification
Rigorous PCR screening using external primers to confirm the replacement of the target gene with the deletion scar/cassette.
Sequencing Confirmation
Sanger sequencing of the entire deletion junction to guarantee precision and confirm the exact sequence of the final modification.
A validated, stepwise procedure ensuring stable genomic modification.
Deletion Cassette Design & Synthesis
Competent Cell Transformation
Selection & Primary Verification
Marker Curing & Final QC
Design: Determination of homology arm size and selection of the appropriate selection marker.
Synthesis: Fabrication of the linear DNA cassette ready for recombination.
Competence: Induction of natural competence in the B. subtilis host strain.
Transformation: Introduction of the linear deletion cassette into competent cells.
Selection: Isolation of colonies showing resistance to the selection marker (indicating successful recombination).
Verification: Initial PCR screening to confirm the gene replacement event.
High Efficiency in B. subtilis
Leveraging B. subtilis's natural competence for highly efficient homologous recombination-based gene replacement.
Protease-Free Chassis
Expertise in sequential deletion of major secreted protease genes, ideal for enhancing the yield of secreted recombinant proteins.
Marker-less Option
Ability to remove the selection marker cleanly for industrial strains and to enable subsequent iterative Gene Knock-out procedures.
Guaranteed Genetic Stability
All deletions are chromosomally integrated, ensuring genetic stability without the need for plasmid maintenance.
"We needed a B. subtilis strain with a triple protease knockout to stabilize our secreted enzyme. CD Biosynsis delivered the strain with all three genes precisely deleted and fully verified."
Dr. Samuel Liu, R&D Director, Industrial Enzymes
"The marker-less knockout option was essential for our regulatory pathway. Their counter-selection protocol for marker removal was highly effective, leaving us with a clean genome."
Ms. Janet Chen, Lead Bioengineer, Food Ingredients
"The precise deletion of a metabolic shunt gene successfully diverted 20% more carbon flux toward our target amino acid. The process was fast and the sequencing data was perfect."
Dr. Kenji Tanaka, Principal Scientist, Fermentation
"We appreciate the clear PCR and sequencing confirmation steps after each gene deletion. It gave us high confidence in the genetic integrity of the final chassis strain."
Mr. Alex Johnson, Research Manager, Biologics Production
"Their expertise in inducing and leveraging B. subtilis competence was key. They achieved a high transformation and knockout rate for a traditionally difficult-to-edit gene."
Dr. Maria Gomez, Group Leader, Synthetic Biology
Why is B. subtilis highly amenable to gene knockout?
B. subtilis naturally develops competence, allowing it to efficiently take up linear exogenous DNA. This facilitates homologous recombination, where the DNA cassette with flanking homology arms can directly replace the target genomic sequence.
What are the key applications of B. subtilis knockout strains?
Primary applications include eliminating native proteases (for stable protein secretion), blocking competing metabolic pathways (for Pathway Optimization), and constructing defined chassis strains for Synthetic Biology.
Do you offer marker-less gene knockout?
Yes. We can remove the temporary antibiotic selection marker after successful knockout using techniques like counter-selection (e.g., sacB gene) or site-specific recombinases (e.g., Cre/Lox), allowing for subsequent Multi-Gene Knockout.
How do you verify the deletion is correct?
Verification is done through both PCR (to confirm the change in locus size) and Sanger sequencing (to confirm the exact sequence of the deletion junction, including the scar/residual sequence).
How much does Metabolic Engineering services cost?
The cost of Metabolic Engineering services depends on the project scope, complexity of the target compound, the host organism chosen, and the required yield optimization. We provide customized quotes after a detailed discussion of your specific research objectives.
Do your engineered strains meet regulatory standards?
We adhere to high quality control standards in all strain construction and optimization processes. While we do not handle final regulatory approval, our detailed documentation and compliance with best laboratory practices ensure your engineered strains are prepared for necessary regulatory filings (e.g., GRAS, FDA).
What to look for when selecting the best gene editing service?
We provide various gene editing services such as CRISPR-sgRNA library generation, stable transformation cell line generation, gene knockout cell line generation, and gene point mutation cell line generation. Users are free to select the type of service that suits their research.
Does gene editing allow customisability?
Yes, we offer very customised gene editing solutions such as AAV vector capsid directed evolution, mRNA vector gene delivery, library creation, promoter evolution and screening, etc.
What is the process for keeping data private and confidential?
We adhere to the data privacy policy completely, and all customer data and experimental data are kept confidential.
CRISPR-Cas9 technology represents a transformative advancement in gene editing techniques. The main function of the system is to precisely cut DNA sequences by combining guide RNA (gRNA) with the Cas9 protein. This technology became a mainstream genome editing tool quickly after its 2012 introduction because of its efficient, simple and low-cost nature.
The CRISPR gene editing system with its Cas9 version stands as a vital instrument for current biological research. CRISPR technology enables gene knockout (KO) through permanent gene expression blockage achieved by sequence disruption. Various scientific domains including disease modeling and drug screening employ this technology to study gene functions. CRISPR KO technology demonstrates high efficiency and precision but requires confirmation and verification post-implementation because unsatisfactory editing may produce off-target effects or incomplete gene knockouts which impact experimental result reliability. For precise and efficient Gene Editing Services - CD Biosynsis, Biosynsis offers comprehensive solutions tailored to your research needs.
The CRISPR-Cas9 knockout cell line was developed using CRISPR/Cas9 gene editing to allow scientists to remove genes accurately for research on gene function and disease models and pharmaceutical discovery. Genetic research considers this technology essential due to its high efficiency together with simple operation and broad usability.
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CD Biosynsis is a leading customer-focused biotechnology company dedicated to providing high-quality products, comprehensive service packages, and tailored solutions to support and facilitate the applications of synthetic biology in a wide range of areas.