CRISPR & Homologous Recombination

Bacillus subtilis Genome Editing & Metabolic Engineering Solutions

We deliver custom-engineered Bacillus subtilis strains with sequence-verified genome edits and optimized metabolic pathways for your production goals.

Custom gRNA and donor template design for targeted edits
Double-strand break or recombinase-mediated integration
Sequence-validated clonal isolates with phenotypic data

Start Your Strain Engineering Project

Engineer B. subtilis with Confidence

Our integrated design-build-test workflow combines CRISPR-Cas9 or homologous recombination with systematic screening to deliver stable, characterized strains for enzymes, metabolites, or therapeutic proteins.

Target locus selection and editing strategy design
Transformation and marker-based selection
Colony PCR and Sanger sequencing verification
Shake-flask cultivation and product quantification

Sequence-Verified Clones

Custom Pathway Optimization

QC Data Package

Our Approach

Systematic Design-Build-Test for B. subtilis

We combine rational design, efficient transformation, and robust screening to deliver edited strains with confirmed genomic modifications and desired production characteristics.

Design & Build

We design gRNAs and donor templates based on your target genes and pathway goals. Constructs are assembled and transformed into B. subtilis using electroporation or natural competence, with antibiotic or auxotrophic selection.

Target analysis and editing strategy
GRNA and donor template construction
Transformation and selection of edited clones

Test & Characterize

Primary clones are screened by colony PCR and Sanger sequencing to confirm the intended edit. Positive isolates are further assessed for growth, product yield, and genetic stability under defined conditions.

Colony PCR and sequence verification
Shake-flask cultivation and product analysis
Data package with genotype and phenotype metrics

Custom Editing Strategy

We tailor the editing approach—CRISPR-Cas9, homologous recombination, or markerless methods—based on locus, strain background, and project requirements.

Efficient Transformation & Screening

Optimized B. subtilis transformation protocols and high-throughput colony screening reduce timelines and increase success rates for complex edits.

Iterative Optimization Cycles

For metabolic engineering, we apply iterative rounds of editing–screening–analysis to fine-tune pathway flux and product titers as needed.

Deliverables

What You Receive

Each project delivers a defined set of engineered strains plus a comprehensive QC and characterization report.

Standard Deliverables

Engineered B. subtilis clone(s) – cryopreserved glycerol stocks (≥2 vials per strain)
Sequencing data confirming the intended edit(s) at the target locus
Shake-flask cultivation data (growth curve, product titer or activity where applicable)
Final project report with methods, results, and quality control.

Additional When Scoped

Parental control strains and plasmids/vectors used
Off-target analysis via targeted amplicon sequencing (as agreed in scope)
Metabolic pathway modeling or flux analysis summary
Scale-up recommendations or process development report

Service Scope

Tailored to Your Project

The table below outlines typical parameters that are defined during the project scoping phase. All details are confirmed in the statement of work.

Parameter	Typical project scope
Editing Modality	CRISPR-Cas9 knockout, base editing, or markerless homologous recombination; knock-in and precise edits evaluated case by case
Target Locus	Any defined genomic locus in B. subtilis 168, WB800, or other user-provided strains; scope confirmed during design review
Transformation Method	Electroporation or natural competence; protocol optimized per strain background
Selection Strategy	Antibiotic resistance (e.g., chloramphenicol, kanamycin, spectinomycin) or auxotrophic complementation; marker removal available when scoped
Screening Depth	Scoped per project after consultation
Phenotypic Characterization	Shake-flask cultivation under defined conditions; product titer or enzyme activity measurement (1–2 time points). Additional analytics (HPLC, LC-MS) as scoped
Off-Target Assessment	In silico off-target prediction; optional targeted amplicon sequencing of top predicted sites (scope and cost agreed per project)
Genetic Stability	PCR and sequencing of isolated clones after serial passaging (number of generations agreed in scope)
Project Timeline	Scoped per project after consultation
Technical Support	Named scientific contact at project start; milestone review calls; email response within 1 business day

Workflow

From Design to Deliverable in Five Phases

Our structured workflow ensures transparent communication and reproducible results at every stage of your B. subtilis engineering project.

Project Scoping & Design

We review your target gene(s), desired edits, and production goals. Together we define editing strategy, construct design, screening methods, and acceptance criteria.

Construct Assembly

GRNAs (if CRISPR-Cas9), donor templates, and any required plasmids are designed, synthesized, and assembled. Reagents are sequence-verified before transformation.

Transformation & Selection

Constructs are transformed into the chosen B. subtilis strain. Transformed cells are plated on selective media; colonies are picked for primary screening.

Genotype Confirmation

Primary clones are analyzed by colony PCR. PCR products are cleaned and Sanger-sequenced to confirm the intended genome edit. At least 2–3 verified isolates are progressed.

Characterization & Delivery

Verified clones are cultivated in shake flasks under defined conditions. Growth and product metrics are measured. A final report with all data and glycerol stocks is delivered.

Applications

Where Engineering B. subtilis Adds Value

We support a wide range of industrial and research applications that benefit from precise genome editing in this robust Gram-positive host.

Pathway Engineering for High-Value Biochemicals

Evaluate flux toward vitamins (e.g., menaquinone-7), organic acids, amino acids, or other small molecules by knocking out competing pathways and overexpressing bottleneck enzymes.

Gene knockout to eliminate byproduct pathways
Integration of heterologous biosynthesis genes
Iterative cycle to balance growth and production

Heterologous Protein and Enzyme Engineering

Secretion of industrial enzymes, therapeutic proteins, or functional peptides. Editing can evaluate secretion efficiency, reduce proteolysis, or remove endogenous impurities.

Genomic integration of expression cassettes
Protease knockout to evaluate product stability
Shake-flake evaluation of yield and activity

Chassis Strain Refinement

Construct minimal-genome or optimized chassis strains by deleting non-essential genomic regions, inserting synthetic circuits, or streamlining metabolism for predictable performance.

Large deletion or devitalized region removal
Insertion of biosensors or regulatory circuits
Performance validation under process conditions

FAQs

Common Questions About Bacillus subtilis Genome Editing

We address typical concerns regarding scope, timelines, and deliverables for our B. subtilis engineering services.

We commonly work with B. subtilis 168 (and its derivatives such as WB800, WB600) and other research strains. If you have a proprietary or non-standard strain, we will evaluate its transformability and editing compatibility during the scoping phase.
Primary screening uses colony PCR across the edited region, followed by Sanger sequencing of the entire modified locus. For knock-ins, we also confirm junction sequences. Off-target analysis is performed in silico and can be extended with targeted amplicon sequencing if scoped.
Each delivered clone includes a certificate of analysis with sequencing chromatograms, alignment to the designed edit, cultivation data (growth curve and product titer where applicable), and a summary of methods and materials used.
Yes. We support iterative design–build–test cycles for multi-locus modifications. Each cycle includes genotypic verification and shake-flask phenotyping. The number of cycles and scope of analytics are defined in the project SOW.
A single-locus knockout with sequencing and shake-flask characterization typically completes a timeline confirmed during project scoping. Multi-locus or pathway engineering projects require additional time depending on the number of edits and complexity of analysis.
In silico off-target prediction is included as standard. If requested, we can perform targeted amplicon sequencing of the top predicted off-target sites (scope and cost discussed during project scoping).

Scientific Literature

References

Lü X, Wu Y, Lin L, et al. [Strategies and tools for metabolic engineering in Bacillus subtilis]. Sheng wu gong cheng xue bao = Chinese journal of biotechnology. 2021;37(5):1619-1636. View on PubMed
Wu S, Sun X, Fan T, et al. Metabolic Engineering Strategy for Bacillus subtilis Producing MK-7. Foods (Basel, Switzerland). 2025;14(23). View on PubMed
Loubat A, Wolfender C, Calabre M, et al. Advancing Fast-Track Genome Engineering in Bacillus subtilis Phages. ACS synthetic biology. 2026;15(2):687-700. View on PubMed
Cao X, Wang X, Chen R, et al. Improving Bacillus subtilis as Biological Chassis Performance by the CRISPR Genetic Toolkit. ACS synthetic biology. 2025;14(3):677-688. View on PubMed
Liu ZY, Yu XZ. Engineering Bacillus subtilis for high-value bioproduction: recent advances and applications. Microbial cell factories. 2025;24(1):182. View on PubMed

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