Custom Microbial Strain Engineering

Vibrio natriegens Multi-Gene Knockout Strain Construction

Markerless, sequential or multiplexed knockout strains of Vibrio natriegens built using CRISPR-Cas9 and natural transformation.

Markerless genome editing — no antibiotic markers retained
Sequential or multiplexed rounds for multi-gene deletion
Verified clonal strains delivered with full genotyping data

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Fast-Track Your Pathway Engineering

Leverage Vibrio natriegens' rapid doubling time and natural competence for streamlined construction of defined knockout strains, supporting metabolic engineering and synthetic biology projects from design to validated clones.

GRNA design and off-target in silico assessment
Plasmid-based Cas9 and gRNA delivery system
Natural transformation with linear repair templates
PCR and Sanger sequencing confirmation of each deletion

Markerless Editing

Fast Growth Host

Customizable Scope

Our Approach

Markerless Knockout via NT-CRISPR

We combine CRISPR-Cas9 with Vibrio natriegens' natural transformation pathway to achieve precise, scarless gene deletions without antibiotic resistance markers. The workflow is tailored to your target genes and can be extended to multiple rounds or simultaneous multiplexed edits.

Design & Build

Our bioinformatics team designs single or multiplex gRNAs targeting your genes of interest, evaluates off-target potential, and assembles Cas9-expressing plasmids with corresponding repair templates.

GRNA design per target locus
Plasmid construction (Cas9 + gRNA)
Linear repair template design

Transformation & Screening

V. natriegens cells are transformed with the editing plasmids plus repair templates via natural transformation. Edited clones are selected by transient resistance and screened by colony PCR for the desired deletion.

Natural transformation protocol optimized for V. natriegens
Transient selection for edited populations
Colony PCR screening for deletion events

Scalable Multi-Gene Knockout

Sequential rounds or co-transformation of multiple gRNAs enable deletion of two or more genes in the same strain, depending on project scope and target compatibility.

Markerless & Scarless

Editing leaves no exogenous antibiotic marker in the genome, suitable for metabolic engineering where marker-free strains are required for downstream applications.

Full Genotypic Verification

Every candidate clone is verified by PCR spanning the deletion junctions and Sanger sequencing to confirm the intended knockout sequence.

Deliverables

What You Receive

Each project delivers a set of verified clonal strains and supporting documentation for seamless integration into your research pipeline.

Standard Deliverables (included in scope)

1–5 verified clonal strains per target knockout (quantity scoped per project)
Glycerol stocks of each confirmed clone
PCR and Sanger sequencing data confirming deletion
Final project report with experimental details

Optional Add‑Ons (when selected)

Additional clones beyond standard number
Whole-genome sequencing for off-target assessment (scoped)
Phenotypic characterization (growth, viability, production)
Plasmid constructs and sequence files

Service Scope

Project Customization

Each project is scoped in collaboration with our scientific team to match your specific target list, timeline, and quality requirements.

Parameter	Typical project scope
Target Organism	Vibrio natriegens (wild-type or derivative strains; custom strains by arrangement)
Editing Method	CRISPR-Cas9 with natural transformation (NT-CRISPR); markerless deletion
Number of Genes	1–5+ genes; sequential or multiplexed depending on gRNA compatibility
Design Support	In silico gRNA design and off-target assessment included prior to construction
Verification	PCR genotyping and Sanger sequencing of each deletion junction
Quality Control	Confirmation of intended deletion; no wild-type contamination; glycerol stock viability check
Technical Support	Named scientific contact at project start; milestone review calls; email response within 1 business day

Workflow

From Gene Targets to Verified Strains

A streamlined five‑step process ensures transparent communication and high‑quality results at every stage.

Project Consultation & Design

Our team reviews your target gene list and strain background, performs in silico gRNA design, and proposes an editing strategy. You approve the design before construction begins.

Plasmid & Repair Template Construction

We build the Cas9‑expressing vector and gRNA expression cassette(s), along with linear repair templates for homologous recombination. All constructs are sequence‑verified.

Natural Transformation & Screening

V. natriegens is transformed with editing plasmids and repair templates. Transformants are screened by colony PCR to identify clones carrying the desired deletion. Multiple rounds are performed for sequential multi‑gene knockout.

Genotypic Confirmation

Positive candidates are purified to clonal populations. Genomic DNA is extracted, and deletion junctions are amplified and Sanger‑sequenced. Only clones with confirmed deletion proceed to final delivery.

Strain Production & Handoff

Verified clones are prepared as glycerol stocks, labelled, and shipped with a project report including methods, QC, and datasets including all design files, PCR images, sequencing chromatograms, and QC results.

Common Applications

Why Knock Out Genes in V. Natriegens?

Vibrio natriegens is valued for its exceptionally fast growth and natural competence, making it an attractive host for metabolic engineering and synthetic biology.

Pathway Redirection

Knock out competing metabolic pathways or byproduct‑forming genes to redirect flux toward a target product (e.g., value‑added chemicals, biofuels).

Eliminate carbon sinks and competing pathways
Combine with heterologous pathway integration
Scoped to your production strain and target

Gene Function Studies

Investigate gene essentiality and phenotype contribution by systematically deleting candidate genes and assessing growth, stress response, or metabolic changes.

Single or combined gene deletions
Comparison with wild‑type controls
Designed to support genetic interaction mapping

Chassis Engineering

Simplify the genome or remove problematic elements (e.g., prophages, non‑essential genes) to create a more predictable and stable synthetic biology chassis.

Target selection guided by literature and your design
Markerless deletions for clean backgrounds
Scalable to multiple loci per project

Frequently Asked Questions

Addressing Common Project Concerns

Find quick answers about scope, timeline, QC, and deliverables below.

Projects typically cover 1–5 target genes, depending on their genomic arrangement and gRNA compatibility. Sequential rounds allow additional deletions, while multiplexed co‑transformation can combine 2–3 genes in one step if design constraints permit. Final scope is determined during consultation.
Yes. Our NT‑CRISPR workflow uses transient selection to enrich for edited cells, but the final strain carries no antibiotic resistance marker in its genome. The deletion is precisely scarless, limited to the intended target sequence.
All candidate clones are confirmed by PCR spanning the deletion region and Sanger sequencing to investigate exact edit. We deliver only clones with confirmed deletion. Glycerol stocks are tested for viability. Off‑target analysis can be included as an optional add‑on, scoped per project.
We can accept custom strains or those previously engineered, subject to compatibility with our transformation protocol. Please discuss your strain background during the consultation so we can assess feasibility and any required modifications.

Scientific Literature

References

Faber A, Politan RJ, Stukenberg D, et al. Expanding genetic engineering capabilities in Vibrio natriegens with the Vnat Collection. Nucleic acids research. 2025;53(13). View on PubMed
Yin M, Ye B, Jin Y, et al. Changes in Vibrio natriegens Growth Under Simulated Microgravity. Frontiers in microbiology. 2020;11:2040. View on PubMed

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