Microbiosci is a leading biotechnology company offering the best E.coli 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 E.coli genome editing services.
Escherichia coli is one of the most extensively studied bacteria that becomes an instrumental model system for the understanding of a plethora of gene functions and regulations in both prokaryotes and eukaryotes. In addition, it’s a very versatile host for the production of heterologous proteins and their mass-production in industrial fermentation systems. E.coli also plays an invaluable role in modern biological engineering and industrial microbiology. More applications are expected to be developed due to the modern molecular technique of the genome editing.
Our E.coli genome editing services based on CRISPR/Cas9 technology and homologous recombination technique. The state-of-art E.coli editing system helps you successfully achieve gene knockout, gene insertion and point mutation for either research or industrial purposes.
E.coli Genome Editing Based on Homologous Recombination
Red/ET Recombination permits the engineering of DNA in E.coli 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 E.coli 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 bacterium. 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
• Homology arms design and suicide plasmid construction
CRISPR/Cas9-mediated E.coli 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 E.coli. 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) or CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy to accomplish the E.coli 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. CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome. (Su, et al. 2016)
Fig.3. Workflow of CRISPR /Cas9-mediated E.coliGenome Editing
• sgRNA design and construction
The advantages of CRISPR based E.coli genome editing in Microbiosci:
• Fast turnaround time
• Scarless genome editing
• Multigene editing: can knock-out up to 3 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, Microbiosci could provide the most excellent service for E.coli genome editing. Our talent experts are dedicated to edit your E.coli 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.
• Downstream application
• Antibiotics and important industrial enzymes production
• Gene function discovery
• Optimize metabolic pathways, increase metabolite production, and achieve industrial production
If you have any special requirements in our E.coli genome editing service, please feel free to contact us at firstname.lastname@example.org or 1-631-626-9181. We are looking forward to working together with your attractive projects.
1. Su, T. et al. A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome. Sci. Rep. 6, 37895; doi: 10.1038/srep37895 (2016).
2. 1Zerbini, F., Zanella, I., Fraccascia, D., König, E., Irene, C., Frattini, L. F., et al. (2017) Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli. Microbial cell factories, 16, 68.
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.