Custom Base Editing in Pseudomonas putida

Pseudomonas putida Base Editing Service

We provide targeted single-nucleotide base editing in Pseudomonas putida using CRISPR-guided deaminase systems to enable precise genomic modifications.

Targeted C→T or A→G base editing in Pseudomonas putida strains
Guide RNA design and base editor plasmid construction included
Sanger sequencing verification of edited clones delivered

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How It Works

Our base editing service delivers validated, sequence-confirmed edited clones of Pseudomonas putida for your metabolic engineering or synthetic biology projects.

Submit strain glycerol stock and target locus information
Guide RNA and base editor design based on your sequence
Transformation, selection, colony PCR screening.

Cytidine or adenine deaminase systems

No double-strand breaks required

Project‑specific QC criteria

Our Approach

Our base editing service for Pseudomonas putida uses CRISPR-guided deaminase fusions to introduce targeted single-nucleotide substitutions at genomic loci defined by the user. The system is delivered as a plasmid or integrative vector carrying a guide RNA and a cytidine or adenine deaminase fused to a Cas9 nickase. Because base editing does not rely on double-strand breaks or homologous recombination, it reduces the risk of unwanted genomic rearrangements and simplifies the screening workflow for point mutations.

Deliverables

What You Receive

After project completion, you receive validated, sequence‑confirmed base‑edited Pseudomonas putida clones along with project documentation of the editing process.

Clone Deliverables

≥1 sequence‑verified edited clone (quantity scoped per project)
Glycerol stock of each confirmed clone
Parental strain control (when included)

Data and Reports

Sanger sequencing chromatograms and alignment files for target locus
In silico off‑target prediction report
Project report summarizing guide design, screening, and validation

Scope of Service

Project Parameters Defined per Case

Each base editing project is scoped individually; the table below outlines typical parameters that are customized after consultation.

Parameter	Typical project scope
Strain	Pseudomonas putida (e.g., KT2440); other strains evaluated during design
Target Locus	Single genomic site; window size and base identity evaluated in silico
Base Editor Type	Cytidine deaminase (C→T) or adenine deaminase (A→G) – selected per desired conversion
Delivery Vector	Plasmid or integrative vector; scoped for stability and marker availability
Screening Method	Antibiotic selection followed by colony PCR; Sanger sequencing confirmation
Quality Control	Sanger sequencing of target region; in silico off‑target prediction; optional targeted amplicon sequencing (as scoped)

Workflow

From Strain to Validated Clone

Our base editing service follows a streamlined five‑step process that covers design, construction, transformation, screening, and final validation.

Strain Submission and Target Selection

You provide a glycerol stock of your target Pseudomonas putida strain and the genomic locus (sequence coordinates) for base editing.

Guide RNA and Base Editor Design

We design guide RNAs targeting your locus and select the appropriate base editor system (cytidine or adenine deaminase) for the desired nucleotide change.

Plasmid Construction and Transformation

Guide RNA and editor sequences are cloned into a delivery plasmid, sequence‑verified, and then introduced into your Pseudomonas putida strain via electroporation or conjugation.

Selection and Colony Screening

Transformants are plated on selective medium; individual colonies are picked and screened by colony PCR to identify those containing the desired edit.

Sequencing Verification and Clone Delivery

Putative edited clones are confirmed by Sanger sequencing across the target window; confirmed clones are prepared as glycerol stocks and delivered with full sequencing data.

Applications

Build Genetic Circuits

Create precise knock‑in of point mutations to tune genetic parts (ribosome binding sites, terminators) for reliable circuit performance.

Introduce or remove restriction sites for DNA assembly
Fine‑tune gene expression levels without deleting open reading frames
Combine with other editing modalities as scoped per project

Frequently Asked Questions

Common Questions About Pseudomonas putida Base Editing

We address typical scope, validation, and technical questions to help you plan your project.

We routinely work with Pseudomonas putida KT2440 and other commonly used lab strains. Isolates or engineered variants can be evaluated during the project scoping phase for compatibility with our base editor delivery systems.
We support cytidine deaminase (C→T) and adenine deaminase (A→G) base editing. The service is designed for single‑nucleotide substitutions within an editable window determined by the guide RNA and deaminase fusion. The target locus is evaluated in silico before design.
Each candidate clone is screened by colony PCR followed by Sanger sequencing of the target locus. The sequencing data is aligned to the reference sequence and the editing efficiency is reported. Off‑target sites are predicted in silico; targeted amplicon sequencing can be added as a scoped option.
Multiplex base editing (simultaneous editing of several loci) is possible but requires separate scoping due to increased complexity in guide design, transformation, and screening. We recommend discussing your multiplex goals during the initial consultation.
Project timelines are defined during scoping and depend on the number of targets, strain characteristics, and required validation depth. Typical durations are discussed during the project consultation phase.

Scientific Literature

References

Velázquez E, de Lorenzo V. AND Logic Based on Suppressor tRNAs Enables Stringent Control of Sliding Base Editors in Pseudomonas putida. ACS synthetic biology. 2024;13(12):4191-4201. View on PubMed
Algar E, Al-Ramahi Y, de Lorenzo V, et al. Environmental Performance of Pseudomonas putida with a Uracylated Genome. Chembiochem: a European journal of chemical biology. 2020;21(22):3255-3265. View on PubMed
Weimer A, Kohlstedt M, Volke DC, et al. Industrial biotechnology of Pseudomonas putida: advances and prospects. Applied microbiology and biotechnology. 2020;104(18):7745-7766. View on PubMed
Mueller J, Willett H, Feist AM, et al. Engineering Pseudomonas putida for improved utilization of syringyl aromatics. Biotechnology and bioengineering. 2022;119(9):2541-2550. View on PubMed
Volke DC, Calero P, Nikel PI. Pseudomonas putida. Trends in microbiology. 2020;28(6):512-513. View on PubMed

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