Microbiosci is a leading biotechnology company offering the best Bacillus subtilis 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 Bacillus subtilis genome editing services.
Bacillus subtilis is a well-characterized gram-positive bacterium that has been widely used for the production of heterologous proteins. B. subtilis and some of its close relatives have excellent protein secretory capability and are generally recognized as safe (GRAS), making them important hosts for the production of antibiotics, medicinal proteins, and industrial enzymes.
Our B. subtilis genome editing services based on CRISPR/Cas9 technology and homologous recombination technique. The state-of-art B. subtilis editing system helps you successfully achieve gene knockout, gene insertion and point mutation for either research or industrial purposes.
Bacillus subtilis Genome Editing Based on Homologous Recombination
Red/ET Recombination permits the engineering of DNA in B. subtilis 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 B. subtilis 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 Bacillus subtilis 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 Bacillus subtilis. 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.
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 Bacillus subtilis Genome Editing
• sgRNA design and construction
The advantages of CRISPR based B. subtilis genome editing in Microbiosci:
• 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, Microbiosci could provide the most excellent service for B. subtilis genome editing. Our talent experts are dedicated to edit your B. subtilis 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 B. subtilis 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. Zhang, K. et al. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system. Sci. Rep. 6, 27943; doi: 10.1038/srep27943 (2016)
2. Wu G, Drufva E, Wu K. Fast genome editing in Bacillus subtilis. Eng Life Sci. 2019;19:471–477
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.