CD Biosynsis offers precise Gene Knock-in Services for Pseudomonas putida, a key strategy for introducing novel functions and creating customized microbial chassis for biomanufacturing. Utilizing advanced homologous recombination (HR) mechanisms, often guided by the CRISPR-Cas9 system, we ensure the stable, targeted integration of heterologous genes, entire biosynthetic pathways, or regulatory elements into the P. putida genome. This capability allows for the development of strains with enhanced production yields, tolerance to toxic compounds, or the ability to synthesize high-value chemicals, moving beyond simple deletion to true functional engineering.
Get a QuoteTargeted gene knock-in is the cornerstone of metabolic pathway construction, enabling the stable expression of foreign genes within the microbial host. Unlike plasmid-based expression, genomic integration ensures long-term genetic stability, prevents plasmid loss under non-selective conditions, and reduces the metabolic burden often associated with high copy number plasmids. Our services focus on integrating DNA fragments—ranging from single genes to complex pathways—into safe harbor loci (e.g., non-essential intergenic regions or specific neutral sites) of the P. putida genome, guaranteeing predictable and sustainable expression of the new genetic circuit for industrial applications.
Large Fragment Knock-in
Integration of DNA fragments up to 10 kb or more, containing operons or entire heterologous metabolic pathways (e.g., for terpene or polyketide synthesis).
Safe Harbor Integration
Targeting neutral genomic sites to ensure high expression without negatively impacting cell growth or essential host functions.
Multi-Copy Integration
Targeting multiple low-copy loci simultaneously to achieve optimized gene dosage and maximal product yield.
Fluorescent Reporter Knock-in
Integration of reporter genes (e.g., GFP, mCherry) under specific promoters to monitor gene expression and cellular pathway activity.
Protein Tagging (C- or N-terminal)
Precise knock-in of affinity tags (e.g., His-tag, Strep-tag) or localization signals onto native P. putida proteins.
Single Nucleotide Polymorphisms (SNPs)
Introduction of precise point mutations for directed evolution studies or to alter enzyme kinetics and regulatory feedback mechanisms.
Promoter/Terminator Swapping
Replacing native regulatory elements with stronger or inducible parts to achieve precise control over gene expression levels.
Our integrated approach utilizes the precision of CRISPR/Cas9 to guide high-efficiency homologous recombination.
1. Design & Donor Template Prep
2. Editing Component Delivery
3. Selection & Screening
4. Validation & Final Strain Curing
Selection of the genomic integration site and desired regulatory elements.
Design of Cas9 sgRNA targeting the integration locus.
Synthesis of the donor DNA fragment containing the gene of interest flanked by optimized homology arms.
Assembly of the CRISPR-Cas9 components and donor template into a single delivery system.
Introduction into P. putida via conjugation or electroporation.
Genotype Verification: Full sequencing of the modified locus to confirm precise, single-copy integration without off-target effects.
Plasmid Curing: Removing temporary Cas9 and selection plasmids for a marker-free, stable final strain.
Deliver the verified engineered strain and a detailed data package.
CRISPR-Assisted HR
Utilizing Cas9 to create a targeted double-strand break (DSB) at the desired locus, dramatically stimulating the efficiency of homologous recombination (HR).
Markerless Integration
Implementation of selection/counter-selection systems (e.g., sacB or inducible selection) to remove all markers after successful integration, yielding a clean industrial strain.
Multiplex Integration
Simultaneous knock-in of multiple separate genes or pathways into different genomic loci to accelerate complex pathway assembly.
Why is genomic integration better than plasmid expression?
Genomic integration ensures the engineered trait is permanently stable and passed on to all daughter cells without relying on antibiotics or selection pressure. This is essential for long-term, large-scale industrial fermentation.
What determines the size limit for gene knock-in?
The size limit is primarily determined by the capacity of the donor DNA delivery system (e.g., plasmid size) and the efficiency of the homologous recombination machinery. We routinely integrate large fragments up to 10-15 kb, covering entire biosynthetic gene clusters.
What are safe harbor loci?
Safe harbor loci are specific, non-essential regions of the genome identified as optimal places for inserting foreign DNA. Insertion here minimizes disruption to native cellular functions, resulting in a healthier, more robust engineered host.
How do you verify a successful knock-in?
We use a combination of junction PCR (using primers that flank the insertion site and the inserted gene) and full Sanger sequencing of the entire modified region to confirm the precise, error-free integration of the new DNA.
CRISPR-Cas9 technology represents a transformative advancement in gene editing techniques. The main function of the system is to precisely cut DNA sequences by combining guide RNA (gRNA) with the Cas9 protein. This technology became a mainstream genome editing tool quickly after its 2012 introduction because of its efficient, simple and low-cost nature.
The CRISPR gene editing system with its Cas9 version stands as a vital instrument for current biological research. CRISPR technology enables gene knockout (KO) through permanent gene expression blockage achieved by sequence disruption. Various scientific domains including disease modeling and drug screening employ this technology to study gene functions. CRISPR KO technology demonstrates high efficiency and precision but requires confirmation and verification post-implementation because unsatisfactory editing may produce off-target effects or incomplete gene knockouts which impact experimental result reliability. For precise and efficient Gene Editing Services - CD Biosynsis, Biosynsis offers comprehensive solutions tailored to your research needs.
The CRISPR-Cas9 knockout cell line was developed using CRISPR/Cas9 gene editing to allow scientists to remove genes accurately for research on gene function and disease models and pharmaceutical discovery. Genetic research considers this technology essential due to its high efficiency together with simple operation and broad usability.
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CD Biosynsis is a leading customer-focused biotechnology company dedicated to providing high-quality products, comprehensive service packages, and tailored solutions to support and facilitate the applications of synthetic biology in a wide range of areas.