E. coli Gene Knockout Service
OverviewServiceSamplesAdvantagesCase StudyFAQs
Overview
Service Overview
At Creative BioMart Microbe, we provide a mature, stable, and high-success-rate gene knockout service built on well-characterized E. coli strains. Our platform is optimized for the targeted deletion of non-essential genes, ensuring that engineered strains retain normal growth characteristics and basal metabolic integrity.
We employ a dual-platform strategy combining λRed recombineering with CRISPR/Cas-assisted genome editing. This composite approach enables scarless, marker-free deletion at base-pair precision, eliminating residual antibiotic cassettes or loxP scars that could interfere with downstream applications. Whether your goal is metabolic pathway optimization, recombinant protein expression enhancement, or industrial strain development, our streamlined workflow delivers sequence-verified knockout strains with full documentation.
Supported Strains
Our service is validated across the most widely used laboratory and industrial E. coli backgrounds. Additional K-12 and B-lineage strains are available upon request.
- E. coli Nissle 1917
- E. coli MG1655
- E. coli BL21 (DE3)
- E. coli DH5α
Figure 1. Schematic illustration of the λRed-CRISPR/Cas9-based scarless gene knockout workflow in E. coli. The system integrates λRed-mediated homologous recombination with CRISPR/Cas9 counter-selection to achieve marker-free deletion.
Creative BioMart Microbe offers end-to-end microbial genome engineering services, from knockout strategy design to validated strain delivery. Contact us for a custom quote.
Our Service
Service Workflow
Service Details
Single Gene Knockout Service
We perform precise deletion of individual non-essential genes in standard E. coli strains. Each project includes optimized sgRNA or homology arm design, recombination template construction, and dual-layer verification by PCR and Sanger sequencing. Deletion fragments range from 500 bp to 10 kb, and every knockout is confirmed to leave no residual scar sequence when the marker-free option is selected.
Multiplex / Sequential Gene Knockout Service
For projects requiring the removal of multiple genetic targets, we offer sequential multiplex knockout with accumulated expertise across 100+ projects. By iteratively applying our λRed-CRISPR/Cas9 platform, we achieve cumulative deletion of up to 3–5 genes per project cycle while maintaining genomic stability. Multiplex projects include intermediate QC checkpoints to ensure each deletion event is correct before proceeding.
Fragment & Operon Deletion Service
This service targets contiguous genomic regions up to 10 kb, including full operons or competing metabolic branches. Large deletions are designed with extended homology arms to preserve chromosomal integrity and prevent polar effects on adjacent essential genes. Ideal for clearing competing pathways to redirect carbon flux toward target products.
Marker-Free Scarless Knockout Service
Our premium scarless option eliminates all selection markers and heterologous sequences from the final genome. Using transient CRISPR/Cas9 counter-selection coupled with self-excising cassettes or direct homology-directed repair, we restore a clean genomic background indistinguishable from wild-type except for the intended deletion. This option is strongly recommended for industrial host strains and regulatory-sensitive applications.
Service Specifications
Knockout Capability
- Target type: Non-essential genes (essential genes require prior feasibility assessment)
- Deletion size per event: 500 bp – 10 kb
- Multiplex editing: Sequential knockout of multiple targets available
Success Rate
- Single gene knockout: ≥95%
- Multiplex sequential knockout: ≥85%
Turnaround Time
| Project Type |
Timeline |
| Single gene knockout |
2–3 weeks |
| Multiplex knockout (2–3 genes) |
3–6 weeks |
| Multi-site sequential knockout (4–5 genes) |
6–10 weeks |
Timeline may vary based on gene complexity and strain background. Custom quotes available for expedited projects.
Deliverables
- Engineered strain (glycerol stock, two vials)
- Knockout validation report (PDF)
- PCR verification gel images / chromatograms
- Sanger sequencing results (forward and reverse traces)
- Growth curve comparison data (wild-type vs. knockout)
- Detailed protocol summary and strain genotype description
Quality Control
- PCR verification with 100% target-region coverage
- Bidirectional Sanger sequencing confirmation
- Strain purity check (single-colony isolation)
- Growth-neutral validation via growth curve assay
Sample Requirements
| Required Information |
Optional Information |
Not Accepted |
- Target gene name, locus tag, or full nucleotide sequence
- Host strain designation (e.g., MG1655, BL21 (DE3))
- Desired deletion coordinates or gene boundaries
|
- Scarless (marker-free) knockout preference
- Antibiotic marker retention requirement (if not scarless)
- Special culture conditions or selective media needs
|
- Unknown or unannotated gene sequences without prior consultation
- Essential genes without a pre-approved conditional knockout strategy
|
Our Advantages
- High Editing Efficiency: Gene knockout success rate ≥95% for standard non-essential genes, backed by 100+ completed projects across diverse strain backgrounds.
- Multiplex Capability: Support for sequential deletion of up to 3–5 genes per project cycle, enabling complex metabolic engineering with an overall success rate ≥85%.
- Broad Strain Compatibility: Proven expertise in Nissle 1917, MG1655, BL21 (DE3), DH5α, and additional K-12 / B strains upon request.
- Rapid Turnaround: Standard single-gene projects delivered in as fast as 2–3 weeks, with clear milestone updates at every stage.
- Comprehensive QC Package: Deliverables include PCR images, sequencing chromatograms, growth curve data, and a detailed strain report suitable for publication or regulatory submission.
Case Study
Case Study 1: CRISPR/Cas9-Mediated Multiplex Knockout in E. coli BW25113 for Enhanced 5-Aminolevulinic Acid Production
Research in Journal of Biological Engineering demonstrates multiplex CRISPR/Cas9-mediated knockout of six competing pathway genes (hemF, gdhA, ybdK, gadB, dppA, mppA) in E. coli BW25113. Using λRed-assisted recombineering with ~400 bp homology arms, researchers achieved marker-free deletions verified by PCR and sequencing. The engineered strain remained growth-neutral in both LB and minimal media while delivering a 2.42-fold increase in 5-aminolevulinic acid titer, reaching 2,099.7 mg/L. This case illustrates how sequential scarless knockout enables rapid flux redistribution for industrial strain development without residual genomic scars.
Figure 2. The results of all mutant strains with respect to ALA production and growth rates. The ALA production and growth rates of the control D0 strain harboring pET28b-ALA-LAA are also indicated for comparison.
Case Study 2: CRISPR/Cas9-Mediated Knockout of Competing Pathway Genes in E. coli BW25113 for Enhanced Vitamin K2 Production
This research demonstrates CRISPR/Cas9-mediated knockout of competing pathway genes entB and pabC in E. coli BW25113-T7. Using λRed-assisted recombineering with ~400 bp homology arms, researchers achieved marker-free deletions verified by colony PCR. Knockout strains remained growth-neutral in both LB and minimal media. Eliminating entB redirected metabolic flux from enterobactin toward menaquinol synthesis, raising Vitamin K2 titer from 303 mg/L to 724 mg/L. Further overexpression of ispB combined with optimized batch fermentation achieved 1,360 mg/L. This case illustrates how targeted scarless knockout of competing pathways enables high-yield industrial strain development.
Figure 3. VK2 production in CRISPR/Cas9-engineered E. coli mutant strains with entB and pabC knockouts.
FAQs
Q: Can essential genes be knocked out?
A: We generally do not recommend knocking out essential genes in standard laboratory strains, as this typically results in lethality. For essential targets, we can discuss alternative strategies such as conditional knockouts, regulated expression systems, or hypomorphic alleles upon project consultation.
Q: Is the knockout guaranteed to be growth-neutral?
A: For validated non-essential genes, we provide growth curve comparison data (wild-type versus knockout) to demonstrate that the deletion does not impair normal growth or biomass accumulation under standard laboratory conditions.
Q: Do you support simultaneous knockout of multiple genes?
A: Yes. We offer sequential multiplex knockout services. Depending on project complexity, we can delete up to 3–5 genes in a single project cycle with an overall success rate ≥85%. Each intermediate clone is sequence-verified before proceeding to the next target.
Q: Will there be any scar or resistance marker left in the genome?
A: Our marker-free scarless knockout option removes all selection cassettes and residual heterologous sequences (no loxP, no FRT, no antibiotic markers), leaving only the intended deletion. A standard knockout may retain a selection marker unless the scarless upgrade is specified.
Q: What strain background should I provide?
A: We accept standard strains including Nissle 1917, MG1655, BL21 (DE3), and DH5α. If you wish to use a custom host strain, please provide complete genotype information and growth parameters so we can assess editing feasibility.
Q: How long does the entire process take?
A: A standard single-gene knockout typically takes 2–3 weeks from target confirmation to strain delivery. Multiplex or sequential projects range from 3–10 weeks, depending on the number of targets and strain-specific editing efficiency.
References:
- Ye, C., et al. (2022). Metabolic engineering of Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification. Journal of biological engineering, 16(1), 26.
- Ye, C., et al. (2026). Metabolic engineering of Escherichia coli BW25113 for the production of Vitamin K2 based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification. Journal of Biological Engineering.
