Bacillus subtilis, Construction of Recombinant Strains (CAT#: LBGF-0126-GF99)
This product utilizes Bacillus subtilis as a robust microbial chassis for the heterologous expression of a wide array of exogenous proteins. Known for its "Generally Recognized as Safe" (GRAS) status and superior protein secretion capabilities, this system is engineered to bypass the limitations of E. coli (such as endotoxin contamination and inclusion body formation). Our platform offers a modular approach, allowing for the customized selection of host strains, vector types, and regulatory elements to meet diverse research and industrial yields.
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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Product Information
Product Overview
This product utilizes Bacillus subtilis as a robust microbial chassis for the heterologous expression of a wide array of exogenous proteins. Known for its "Generally Recognized as Safe" (GRAS) status and superior protein secretion capabilities, this system is engineered to bypass the limitations of E. coli (such as endotoxin contamination and inclusion body formation). Our platform offers a modular approach, allowing for the customized selection of host strains, vector types, and regulatory elements to meet diverse research and industrial yields.
Target
Bacillus subtilis
Applications
1. Drug Discovery: High-throughput screening of antimicrobial peptides (AMPs) or small molecule inhibitors without LPS interference. 2. Synthetic Biology: Construction of multi-stage genetic circuits, logic gates, and "kill-switches" for environmental biosensing. 3. Immunology: Development of mucosal vaccine vectors where B. subtilis acts as both the adjuvant (via TLR stimulation) and the antigen delivery vehicle. 4. Enzymology: Study of directed evolution and protein stability in a natively high-secreting environment.
We offer two primary methods for establishing the genetic blueprint of the recombinant strain: 1. Plasmid-Based Expression: Utilizes high-copy or low-copy shuttle vectors (e.g., pHT01, pBE-S). This is ideal for rapid screening, high-yield production, and cases where high gene dosage is required. 2. Genomic Integrated Form: Employs homologous recombination (typically at the amyE, thrC, or lacA loci) to insert the exogenous gene directly into the B. subtilis chromosome.
Genetic Architecture
Promoter System
Constitutive or Inducible
Selection Marker
Standard Antibiotic Marker or Markerless/Scarless (No resistance genes)
Delivery & Service Standards
Deliverables
Glycerol Stocks
Shipping Conditions
Dry Ice Shipping
Lead Time
4-8 weeks
Documents
Certificate of Analysis (COA) or Project Report
Verification
PCR Sequencing
Engineered Strain Recovery & Handling
Immediate Post-Delivery Steps
1. Transfer to Storage: Immediately transfer the cryovials to a -80°C ultra-low temperature freezer. 2. Avoid Fluctuations: Do not store the vials in a "frost-free" freezer, as the temperature cycles can damage the cell membranes of the engineered strains.
Strain Recovery Protocol
To ensure maximum viability, follow this standard recovery procedure: 1. Preparation: Prepare the appropriate selective agar medium (e.g., LB agar + specific antibiotic as listed in the COA). 2. Partial Thawing (The "Scratch" Method): * Place the cryovial on dry ice. Do not thaw the entire vial. Use a sterile inoculation loop or pipette tip to "scratch" a small amount of ice from the surface of the frozen stock. 3. Streaking: Streak the cells onto the selective agar plate to obtain single colonies. 4. Incubation: Invert the plate and incubate at the specified temperature (e.g., 37°C for E. coli or 30°C for S. cerevisiae) for 16-24 hours. 5. Subculturing: Pick a single, well-isolated colony to inoculate into liquid media for your experiments.
Usage Precautions & Best Practices
Antibiotic Pressure
1. Maintenance: For strains containing plasmids or specific resistance markers, always use the correct concentration of antibiotics in both solid and liquid media to prevent plasmid loss or contamination.
2. Markerless Strains: If the strain is a "scarless" integrated knockout, antibiotic pressure is not required for maintenance, but periodic genotypic verification is recommended.
Induction Conditions
1. If your strain uses an inducible promoter (e.g., T7/IPTG or Arabinose), ensure the culture reaches the optimal OD600 (typically 0.6-0.8) before adding the inducer. 2. Verify the optimal induction temperature, as engineered strains often produce better results at lower temperatures (e.g., 16-25°C) to prevent inclusion body formation.
Disposal
All waste (vials, tips, plates) must be autoclaved at 121°C for at least 20 minutes or chemically disinfected before disposal.
