Pseudomonas Genome Editing Service

Creative Biogene is a leading biotechnology company offering the best Pseudomonas genome editing services. With years of experience and expertise in microbial genome editing, our talented scientists will work closely with you to provide any help in Pseudomonas genome editing services.

Pseudomonas species, a large class of gram-negative bacteria, exhibit significant biomedical, ecological, and industrial importance. Pseudomonas, a major human pathogen, is able to cause severe infections in vulnerable patients hospitalized with cystic fibrosis, burns, acquired immunodeficiency syndrome, or cancer. Extensive research has been focused to dissect the molecular basis of the infection mechanisms and to develop novel therapeutic means against Pseudomonas infections. The genetic manipulation in Pseudomonas is extremely urgent.

Our Pseudomonas genome editing services based on CRISPR/Cas9 technology and homologous recombination technique. The state-of-art Pseudomonas editing system helps you successfully achieve gene knockout, gene insertion and point mutation for either research or industrial purposes.

Pseudomonas Genome Editing Based on Homologous Recombination

Red/ET Recombination permits the engineering of DNA in LAB using homologous recombination mediated by phage protein pairs, either RecE/RecT or Reda/Redb. The central step in Red/ET recombination is the crossover step between a targeting construct containing homology arms and the target which can be a gene locus on the LAB chromosome by designing a homologous fusion fragment of the target gene, it is cloned into a suicide vector, and the suicide vector is transformed into the target Pseudomonas. An insertion mutant is selected by antibiotic screening. Under the second round of reverse selection pressure, only the mutation that contain second homologous recombination and the loss of the suicide plasmid can survive. By PCR screening and sequencing, we can obtain the mutant of the strain.

Fig.1. Workflow of homologous recombination genome editing

Even the most demanding tasks can be reduced tofour basic steps:

• Homology arms design and suicide plasmid construction
• Recombineering
• Selection/Screening
• Validation

CRISPR/Cas9-mediated Pseudomonas Genome Editing

CRISPR technology, which derived from the immune system present in bacteria and archaea, is an efficient genome-scale editing tool that has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering. It enables fast and reliable genetic manipulation in LAB. Two components are requested to work: a guide RNA (gRNA), e.g. under an RNA polymerase III promoter, and the nuclear localization tag fused DNA endonuclease, with Cas9 being the most commonly used.

Here we use CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) to accomplish the Pseudomonas genome editing.

When Cas9 protein and gRNA are expressed in bacteria cells, Cas9 introduces DSBs that must be repaired by the cells via non-homologous end joining (NHEJ) or homologous recombination (HR). By supplying a DNA repair template for use in HR, various DNA modifications can be obtained.

Fig.2. Workflow of CRISPR /Cas9-mediated Pseudomonas Genome Editing

Project steps:

• sgRNA design and construction
• Transformation
• Selection/Screening
• Validation

The advantages of CRISPR based Pseudomonas genome editing in Creative Biogene:

• Fast turnaround time
• Scarless genome editing
• Multigene editing: can knock-out up to 5 genes simultaneously
• Easy selection: no selectable marker is required

What we could help?

• Gene disruption, deletion or insertion
• Reporter gene and tag integration
• Promoter fine tuning
• Introduction of point mutations

With years of experience in genome editing field, Creative Biogene could provide the most excellent service for Pseudomonas genome editing. Our talent experts are dedicated to edit your Pseudomonas genome with the greatest chance to succeed. Meanwhile, based on the commitment of prompt communication and on-time reporting, our staffs will ensure a high-efficiency service to meet the strict project timelines.

If you have any special requirements in our Pseudomonas genome editing service, please feel free to contact us at . We are looking forward to working together with your attractive projects.

References
1. Chen W, Zhang Y, Zhang Y, Pi Y, Gu T, Song L, Wang Y, Ji Q. CRISPR/Cas9-based Genome Editing in Pseudomonas and Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species. iScience. 2018 Aug 31;6:222-231.
2. Hmelo, L. R., Borlee, B. R., Almblad, H., Love, M. E., Randall, T. E., Tseng, B. S., et al. (2015) Precision-engineering the Pseudomonas genome with two-step allelic exchange. Nature protocols, 10, 1820.
3. Yu D, et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 2000;97:5978–5983
4. Stovicek V, Holkenbrink C. Borodina I. CRISPR/Cas system for yeast genome engineering: advances and applications. FEMS Yeast Res. 2017;17:fox030.

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