Pseudomonas putida Gene Knockout

Pseudomonas putida Gene Knockout Service

Custom gene knockout services in Pseudomonas putida, from target design and editing to genotype-verified clones.

CRISPR-based and alternative deletion routes scoped to your target locus
In silico puzzle-free design: locus review, guide, and construct planning.
Clone screening and genotype confirmation completed before strain.

Knockout programs scoped case by case

Each Pseudomonas putida knockout project is defined against your target gene, strain background, and downstream use before work begins.

Target gene and locus reviewed during design
Editing route and deletion format agreed at scoping
Colony PCR screening plus junction sequencing

KT2440 and related backgrounds

Markerless options when scoped

Sequence-confirmed clones

Our approach

How Pseudomonas putida knockout projects are delivered

Knockout programs are scoped against your target gene and strain background, then designed, executed, and confirmed before handoff.

Locus-aware knockout design

We start from the target gene and the host background you specify, then plan the deletion strategy around locus context, sequence constraints, and the intended downstream use of the strain.

Target locus and sequence context reviewed before design
Guide and construct design matched to the selected editing route
Essentiality and selection-marker choices flagged during scoping

Editing, screening, and genotype confirmation

Editing constructs are assembled and applied to the agreed Pseudomonas putida background; candidate clones are screened across the deletion junction and confirmed by sequencing before handoff.

Editing construct assembly with sequence checks
Colony PCR screening across the deletion junction
Sanger confirmation of the edited locus before handoff

Design built around the locus

Deletion strategy follows the target gene, its chromosomal context, and the host background you specify rather than a fixed template, so the design fits the actual target.

Case-by-case project scope

Candidate clone counts, screening depth, verification level, and acceptance criteria are fixed in the statement of work before execution begins.

Documentation that travels with the strain

Sequencing files, screening summaries, and construct records are assembled so your internal team can retrace exactly how the knockout was built.

Deliverables

What you receive at project handoff

Handoff contents are confirmed at scoping; the lists below show what a typical Pseudomonas putida knockout project includes.

Strain and material handoff

Edited Pseudomonas putida clone(s) carrying the agreed deletion.
Glycerol stocks of sequence-confirmed clones when included in the project.
Editing plasmid or construct DNA when included in the project scope
Parent or unedited reference strain when requested as part.

Data and documentation

Sequencing reads and alignment files covering the edited locus
Colony PCR screening summary for the candidate clones processed
Construct map and primer reference sheets used during the build
Final project report summarizing design, execution, and QC outcomes

Service scope

Scope parameters defined case by case

Every capability below is confirmed during scoping; the statement of work fixes what is included in your project.

Parameter	Typical project scope
Host strain background	Pseudomonas putida KT2440 and other agreed P. putida backgrounds; related Pseudomonas species evaluated case by case during scoping.
Target gene or region	Single-gene deletions are routine; multi-gene or larger cassette deletions are assessed against locus context during design.
Edit format	Markerless deletion, marker-excision, or selection-marker formats selected case by case based on downstream use of the strain.
Editing route	CRISPR-based editing is the primary route; alternative non-CRISPR deletion routes can be incorporated when the host or target requires them.
Target locus constraints	Copy number, probable essentiality, and sequence context are reviewed during design; repetitive or low-complexity regions are flagged early.
Clone screening depth	Colony PCR screening across the deletion junction followed by Sanger sequencing; additional assessment rounds can be scoped.
Off-target assessment	In silico specificity review of the designed guides is standard; targeted amplicon checks at predicted sites are available when project-scoped.
Strain validation level	Genotype-level confirmation is standard; growth or phenotype assessment is added only when written into the project scope.
Deliverable format	Glycerol stocks, construct DNA, sequencing files, and a project report; the exact handoff list is agreed before execution.
Acceptance criteria	Screening and confirmation criteria are defined jointly in the statement of work, so expectations are fixed before the project starts.

Workflow

A typical knockout engagement

A standard engagement moves from project scoping through to sequence-confirmed clone handoff.

Project scoping and feasibility review

We review your target gene, strain background, and intended use, then agree on the deletion format, screening depth, and acceptance criteria in the statement of work.

Target analysis and edit design

The locus is assessed for context, probable essentiality, and sequence constraints, and the editing strategy plus assessment primers are designed around those findings.

Construct assembly and checks

Editing constructs, guides where a CRISPR-based route is selected, and any selection or counterselection elements are assembled and sequence-checked before use.

Strain editing and recovery

The construct is introduced into the agreed Pseudomonas putida background, edited cells are selected, and candidate colonies are recovered for downstream screening.

Clone screening and handoff

Candidate clones are screened by colony PCR across the deletion junction, confirmed by sequencing of the edited locus, then documented and packaged for handoff.

Applications

Where P. putida knockouts are used

Knockout projects are typically commissioned for the study areas below, with targets and confirmation depth agreed at scoping.

Removing competing pathway genes

Deletions of side-pathway or byproduct-forming genes support investigations into how a chosen host routes carbon. Projects are designed around the genes you specify, with deletion format and confirmation depth agreed at scoping.

Single or multi-gene deletions scoped case by case
Markerless or marked formats matched to downstream use
Junction sequencing completed before strain handoff

Gene-function and essentiality studies

Clean deletion mutants are a common starting point for characterizing what a gene does in Pseudomonas putida. Where a target may be essential, the design can be adapted with conditional or inducible setups when that approach is included in scope.

Locus and probable essentiality reviewed during design
Conditional or inducible strategies when project-scoped
Parent or wild-type reference strains on request

Host and chassis engineering programs

Chassis programs often need a series of deletions built on a common background. Sequence-confirmed clones can be handed off for continued strain construction, with each target gene and confirmation level defined in the statement of work.

Sequential or parallel targets as scoped
Consistent background and documentation across builds
Stocks and sequencing records prepared for handoff

FAQ

Questions buyers ask before scoping

Common questions on host range, confirmation, off-target scope, and project handoff.

Knockout projects are typically run in Pseudomonas putida KT2440, the most widely used engineering background. Other P. putida isolates and related Pseudomonas species are evaluated case by case during scoping, because host genetics, restriction systems, and usable selection markers can change the design.
Candidate clones are first screened by colony PCR across the edited junction, then the deletion is confirmed by Sanger sequencing of the locus. Screening depth and the number of clones verified are written into the project scope, so expectations are fixed before work starts.
Markerless deletion formats can be scoped when the downstream application requires an unmarked strain. Where a selection marker is acceptable, simpler formats may be proposed instead. The choice is agreed during design based on the target locus and the intended use of the strain.
In silico specificity review of the designed guides is part of the design step. Targeted amplicon checks at predicted sites can be incorporated when project-scoped. Genome-wide off-target assays are not part of the standard workflow and would need to be discussed as a separate scope.
Knockout outcomes depend on the target locus and host background, so screening covers multiple candidate clones. If the intended edit is not recovered, we review the design and locus with you and agree on next steps, which may include redesign or an alternative deletion route.
Typical handoff includes sequence-confirmed edited clone(s), glycerol stocks when included, sequencing files, a colony PCR screening summary, and a final project report. Parent controls, construct DNA, and phenotype data are added only when they are written into the project scope.

Scientific Literature

References

Choi KR, Lee SY. Protocols for RecET-based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida. Microbial biotechnology. 2020;13(1):199-209. View on PubMed
Velázquez E, Al-Ramahi Y, Tellechea-Luzardo J, et al. Targetron-Assisted Delivery of Exogenous DNA Sequences into Pseudomonas putida through CRISPR-Aided Counterselection. ACS synthetic biology. 2021;10(10):2552-2565. View on PubMed
Sumi S, Mutaguchi N, Ebuchi T, et al. Light Response of Pseudomonas putida KT2440 Mediated by Class II LitR, a Photosensor Homolog. Journal of bacteriology. 2020;202(20). View on PubMed
Han F, Zhang X, Chen Y, et al. A Simple Allelic Exchange Method for Efficient Seamless Knockout of Up to 34-kbp-Long Gene Cassettes in Pseudomonas. Applied biochemistry and biotechnology. 2024;196(8):5616-5630. View on PubMed
Choi KR, Cho JS, Cho IJ, et al. Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida. Metabolic engineering. 2018;47:463-474. View on PubMed

Ready to Start Your Project?

Get a customized quote for your Pseudomonas putida Gene Knockout Service project. Our experts will respond within 24 hours.

No obligation

24h response

Expert consultation