MyPseudomonas

Precision Gene Deletion Platform for Pseudomonas

Core Technology

A complete platform for gene deletion in Pseudomonas that combines cosmid-based homologous recombination, gusA blue-white selection, and the IMES iterative marker excision system.

Red/ET Recombination

Phage-derived Redα, Redβ, Redγ recombinases catalyse precise exchange between the cosmid target and a linear IMES cassette flanked by only 40 bp homology arms.

gusA Blue-White Screen

β-glucuronidase reporter on the vector backbone. Blue colonies = single crossover (discard). White colonies = double crossover (desired deletion mutant).

φC31 IMES Excision

Modified attachment sites B-CC and P-GG drive ~100% intramolecular excision of the resistance marker

Homology-Directed Integration

Lacking a Pseudomonas origin of replication, the cosmid can only be maintained through homologous recombination, eliminating autonomous plasmid replication.

Visual Double-Crossover Selection

Blue/white screening enables direct visual identification of double-crossover mutants, substantially reducing PCR screening.

Clean Final Genotype

After marker excision and plasmid curing - no resistance genes, no foreign DNA. Only the 48 bp inert scar marks the deleted locus.

Engineering Workflow

From cosmid to marker-free mutant in five steps

Pseudomonas mutants engineering - Cosmid manipulation Pseudomonas transformation Pseudomonas mutants blue-white screen Pseudomonas mutants engineering - marker excision Pseudomonas mutants plasmid curing

The Advantages of Our Platform

01 Zero scar cross-reactivity

RR scars do not recombine with identical RR (0%), B-CC (0%), or P-GG (~0.1%). No large-scale rearrangements at any deletion count.

02 Visual mutant selection

Blue-white screening enables rapid discrimination between single- and double-crossover recombinants, dramatically reducing PCR verification.

03 Easy Gene Targeting

Target any gene using just two 60-mer oligonucleotides, eliminating the need to clone long homology arms.

04 One Primer Pair

A universal primer pair amplifies all our IMES Markers using shared 19-20 bp core sequences.

05 Marker-Free Final Strains

Final strains contain no foreign DNA or antibiotic resistance genes, ensuring a clean genetic background.

06 Iterative Genome Engineering

Redesign only the gene-specific homologous primers while keeping the vector architecture and engineering workflow unchanged.

Products & Services

Genome-Wide Cosmids

Cosmid libraries covering full genomes with tiling overlap.

  • 25–40 kb inserts
  • End-sequenced clones
  • Replicate in E. coli.

Deletion Kits

Complete gene deletion package for your Pseudomonas strain of interest.

  • Cosmid containing the chromosomal region of interest
  • IMES cassette (Apr / Tet)
  • Recombination strain
  • Helper vector for IMES cassette excision
  • Protocol and technical support

Custom Strain Engineering

Complete genome engineering service for your Pseudomonas strain.

  • Target analysis and strategy
  • Cosmid and IMES cassette construction
  • Single or multiple gene deletions
  • PCR and sequencing validation
  • Delivery of verified engineered strains