CD Biosynsis offers specialized Bacillus subtilis Gene Knock-in Services, enabling the stable integration of exogenous genetic material into the host chromosome. B. subtilis is a highly efficient host for gene knock-in due to its natural competence, allowing for the precise, homologous recombination-mediated integration of large DNA fragments, synthetic pathways, or single genes. This service is essential for establishing stable, high-copy expression of heterologous proteins (e.g., enzymes, antibodies), integrating entire biosynthetic pathways (Metabolic Pathway Optimization), and engineering novel cellular functions. We provide complete design-to-delivery solutions, including integration cassette design, selection of optimal neutral loci, high-efficiency transformation, and full genetic verification for stable, long-term expression without plasmid loss.
Get a QuoteChromosomal Gene Knock-in is the gold standard for developing industrial microbial strains, offering superior genetic stability compared to plasmids. In B. subtilis, we leverage its natural competence to insert expression cassettes containing the gene of interest, a constitutive or inducible promoter, and a selection marker, flanked by homologous regions targeting a neutral locus (e.g., amyE, thrC). Our expertise in codon optimization and promoter tuning ensures high-level, stable expression of the integrated gene. This technique is vital for applications requiring long fermentation times, such as the production of enzymes, natural products, or secondary metabolites, where plasmid instability is a major limitation.
Codon Optimization for B. subtilis
Optimization of the heterologous gene sequence to match the B. subtilis codon usage bias for maximum translation efficiency and yield.
Promoter and RBS Tuning
Selection of constitutive or inducible promoters and Ribosomal Binding Sites (RBS) to achieve the desired, stable expression level of the integrated gene.
Homology Arm Design
Precise design of flanking homology arms (up to 1500 bp) targeting highly efficient neutral loci for reliable homologous recombination.
Selection of Integration Site
Targeting neutral sites (e.g., amyE) that minimize disruption to the host's metabolism and ensure stable expression without impacting growth.
High-Efficiency Transformation
Optimization of the competence induction protocol for the specific B. subtilis strain to ensure maximum DNA uptake and recombination rate.
Marker-less Integration Option
Use of specialized counter-selection cassettes to remove the antibiotic resistance marker post-integration, providing a clean production strain.
Large Fragment Knock-in
Expertise in chromosomally integrating multi-kilobase DNA fragments, including entire synthetic operons or complex Metabolic Pathway Optimization modules.
Sequential Multiple Knock-in
Strategy development for the stable, sequential integration of multiple genes into different chromosomal loci for robust co-expression.
Full Genetic Verification
Final strains are verified via PCR and sequencing across the integration site to ensure 100% correct insertion and sequence fidelity.
A precision workflow for stable, single-copy integration.
Integration Cassette Design & Prep
Transformation & Selection
Primary Verification & Curing
Final Sequencing & Delivery
Design: Codon optimization, promoter selection, and design of homology arms flanking the integration locus.
Prep: Synthesis and preparation of the linear DNA integration cassette.
Competence Induction: Preparing the B. subtilis host strain for high-efficiency DNA uptake.
Selection: Isolation of colonies showing successful double-crossover homologous recombination using antibiotic selection.
Verification: PCR screening to confirm the insertion at the correct locus and size change.
Curing: Optional removal of the selection marker using counter-selection.
Superior Genetic Stability
Chromosomal integration eliminates plasmid loss and ensures stable gene expression across long-term fermentation runs.
High Integration Efficiency
Leveraging B. subtilis natural competence for highly successful homologous recombination and gene insertion.
Multi-Gene Integration
Capability to integrate large synthetic pathways or multiple genes into different neutral loci for complex strain engineering.
Codon and Promoter Optimized
Integrated gene sequences are optimized for B. subtilis expression, ensuring maximum soluble protein yield.
"We successfully integrated a three-gene operon for a novel secondary metabolite into the B. subtilis genome. The strain exhibited perfect stability and consistent high yield across all our pilot runs, unlike our previous plasmid system."
Dr. Samuel Liu, R&D Director, Natural Products
"The ability to remove the antibiotic marker after the Gene Knock-in was crucial for our industrial application. The resulting clean genome strain met all our regulatory requirements."
Ms. Janet Chen, Lead Bioengineer, Pharma Production
"The codon optimization and promoter choice for our heterologous enzyme were spot-on. The integrated enzyme expressed at a high level and showed significantly improved stability over the entire fermentation period."
Dr. Kenji Tanaka, Principal Scientist, Enzyme Technology
"Their precise targeting of the amyE locus ensured our integrated gene didn't interfere with the host's growth. The sequencing verification gave us full confidence in the final product."
Mr. Alex Johnson, Research Manager, Strain Development
"We used this service for Metabolic Pathway Optimization, integrating several modules sequentially. The strategy for multiple, stable knock-ins worked flawlessly, accelerating our project timeline."
Dr. Maria Gomez, Group Leader, Synthetic Biology
Why choose chromosomal Knock-in over plasmid expression in B. subtilis?
Chromosomal Knock-in offers far greater genetic stability, especially in large-scale or long-term fermentations, as there is no risk of plasmid loss. It also ensures consistent, single-copy expression levels.
What is a 'neutral locus' and why is it important for Knock-in?
A neutral locus (like amyE) is a genomic site where the insertion or deletion of DNA does not negatively impact the host cell's fitness, growth rate, or core metabolism. Targeting neutral loci ensures the stability and viability of the engineered strain.
What is the largest DNA fragment you can integrate?
B. subtilis is excellent for large integrations. We routinely integrate multi-kilobase DNA fragments, including entire synthetic biosynthetic pathways consisting of several genes, utilizing its highly efficient homologous recombination machinery.
Can you perform multiple sequential gene knock-ins?
Yes. By targeting different neutral loci and using orthogonal or curable selection markers, we can sequentially integrate multiple genes or pathways into the B. subtilis genome.
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.