CRISPR/Cas9 Gene Knock-in for Pseudomonas putida

Pseudomonas putida Gene Knock-in

Custom CRISPR/Cas9-mediated gene knock-in services for stable integration of your gene of interest into the Pseudomonas putida genome.

Custom guide RNA and donor template design for efficient integration
Stable genomic insertion via homologous recombination with selection
Verified engineered strain delivered with sequencing and PCR QC

Submit Gene and Target Details

Stable Genomic Integration in a GRAS/QPS-qualified host strains (use-specific)

Pseudomonas putida is a versatile, safe (GRAS) industrial chassis. Our CRISPR/Cas9 knock-in service precisely inserts your gene of interest at defined genomic loci, enabling stable overexpression or functional studies without plasmid loss.

Target-site selection and homology arm design
Donor template construction with antibiotic/counter-selection marker
PCR and Sanger sequencing validation of integration

Custom Gene Integration

Screening & Genotyping QC

Engineered Strain Delivery

Overview

CRISPR/Cas9-Based Knock-in Workflow

Our service combines target-specific guide RNA design, donor template construction with homology arms, and efficient delivery into P. putida to achieve precise, stable genomic integration.

Guide RNA and Donor Design

We select optimal target sites and design custom guide RNAs together with donor templates carrying your gene of interest flanked by homology arms (typically 500–1000 bp each). All designs are reviewed in silico for specificity and efficiency.

In silico off-target assessment of guide RNAs
Homology arm length optimized for your target locus
Selection marker (antibiotic or auxotrophic) included in donor

Transformation and Integration Screening

Cas9, guide RNA, and donor template are delivered into P. putida cells via electroporation or conjugation. Following selection, we screen colonies by PCR and Sanger sequencing to confirm correct knock-in at the intended genomic site.

Multiple transformants screened (quantity scoped per project)
PCR amplicon sequencing across integration junctions
Negative colony controls included for validation

Custom Strain Background

Service is adaptable to common P. putida strains (e.g., KT2440, DOT-T1E, and other laboratory or environmental isolates) with project-specific optimization of transformation efficiency.

Dual-Marker Options

Choose antibiotic resistance markers (gentamicin, kanamycin, streptomycin) or auxotrophic complementation for marker recycling, depending on your downstream application requirements.

Genotypic and Phenotypic QC

Each knock-in strain is verified by PCR, Sanger sequencing, and optionally by qPCR or whole-genome sequencing (as scoped) to confirm single-copy integration and absence of unintended genomic rearrangements.

Handoff

What You Receive

Every project delivers a verified engineered P. putida strain plus all supporting data and materials to continue your research.

Materials Shipped

Engineered Pseudomonas putida strain (verified knock-in, glycerol stock and culture)
Donor template plasmid (sequence-verified)
Guide RNA expression plasmid or ribonucleoprotein (if used)
Selection marker plasmids (when applicable)

Documentation & QC

Full project report including design rationale, gRNA sequences.
PCR gel images and Sanger sequencing chromatograms of integration.
Strain genotype summary (integration site, copy number assessment)
Pilot phenotypic data (growth, morphology) if scoped

Scope

Service Parameters

The table below outlines typical project specifications. All parameters are customized based on your gene, target locus, and downstream application; final scope is defined in the project SOW.

Parameter	Typical project scope
Host Strain	Pseudomonas putida KT2440 (default); other strains (e.g., DOT-T1E, S12) evaluated on a case-by-case basis
Gene of Interest Size	Scoped per project after consultation
Homology Arm Length	Scoped per project after consultation
Selection Marker	Gentamicin, kanamycin, or streptomycin resistance; auxotrophic complementation (e.g., pyrF) available for marker recycling
Integration Site	Intergenic neutral sites (e.g., attTn7, GG1) or a genomic locus of your choice (if transcriptionally silent region)
Number of Clones Screened	Colonies per project; additional screening scoped as needed
Verification Method	PCR (junction-spanning) + Sanger sequencing of integration sites; optional qPCR for copy number or WGS for whole-genome validation
Deliverables Timeframe	Scoped per project after consultation

Process

Project Workflow

From sequence submission to delivered strain, our five-step process ensures transparent progress and rigorous quality control at each stage.

Target Site & Guide Design

You provide the gene of interest and target locus (or we select an intergenic neutral site). Our team designs one to two guide RNAs in the proximity of the integration site and performs in silico off-target analysis. Approved designs move to plasmid construction.

Donor & gRNA Plasmid Assembly

We clone the gene of interest flanked by homology arms into a donor vector carrying a selection marker. The guide RNA is cloned into a Cas9 expression plasmid. All constructs are sequence-verified before delivery to the next step.

Transformation of P. putida

Competent P. putida cells are co-transformed with the Cas9/gRNA plasmid and the donor template (or delivered as a ribonucleoprotein complex). We evaluate cell preparation and electroporation parameters to evaluate integration efficiency.

Selection & Colony Screening

Transformants are plated on selective media. Individual colonies are picked and screened by colony PCR using primers spanning the integration junctions. Positive hits are confirmed by Sanger sequencing of the entire inserted region.

Strain Validation & Handoff

At least one confirmed knock-in clone is re-streaked to support clonal purity. We perform final PCR and sequencing across the integration site, prepare glycerol stocks, and generate a project report including methods, QC, and datasets. The engineered strain and all materials are shipped.

Use Cases

Applications of P. putida Gene Knock-in

Our service supports a wide range of research and industrial goals where stable genomic expression is required.

Pathway Integration

Insert heterologous biosynthetic genes for production of specialty chemicals, biopolymers, or natural products directly into the genome for stable, marker-free expression.

Polyhydroxyalkanoate (PHA) synthesis genes
Aromatic compound degradation pathways
Terpenoid or flavonoid production cassettes

Gene Overexpression Studies

Integrate a gene of interest under a constitutive or inducible promoter to study gain-of-function phenotypes without plasmid maintenance or copy-number variability.

Constitutive (PgroES, Pj23119) or inducible promoters
GFP/fluorescent fusion tags for localization
His/Strep tags for protein purification

Stable Catabolic Enzyme Expression

Integrate enzymes that degrade recalcitrant pollutants (PCBs, explosives) or catalyze stereo-selective transformations for green chemistry applications.

Cytochrome P450 monooxygenases
Laccases and peroxidases
Dehalogenases and nitrilases

FAQ

Frequently Asked Questions

Answers to common questions about our P. putida gene knock-in service, including scope, QC, and project handoff.

Knock-in efficiency depends on the target locus, homology arm design, and strain background. Under standard conditions, we typically observe project-dependent performance that is verified during development integration among selected colonies. Final success is evaluated during the screening step; if no correct integrants are obtained, we redesign the guide RNA or homology arms at no additional charge (within the original project scope).
Our protocol drives integration via double-crossover homologous recombination, which is inherently stable. In strains where a selection marker is retained, we confirm marker presence. For markerless knock-ins (using counter-selection), we investigate the integration by PCR/sequencing and provide a working glycerol stock. Long-term stability can be assessed as a follow-up service if requested.
Our standard service supports inserts up to approximately 5 kb. For larger clusters, we can design a multi-step integration strategy using serial knock-ins or a two-vector system. Please contact us to discuss feasibility and appropriate timelines for large constructs.
You receive PCR gel images showing specific amplification across both homology arms, Sanger sequencing electropherograms of the entire integrated region, and a summary table of tested colonies. Optional whole-genome sequencing (WGS) or qPCR for copy number assessment can be added to the project scope.

Scientific Literature

References

Calero P, Gurdo N, Nikel PI. Role of the CrcB transporter of Pseudomonas putida in the multi-level stress response elicited by mineral fluoride. Environmental microbiology. 2022;24(11):5082-5104. View on PubMed

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