Bacillus licheniformis Genome Editing Service

Microbiosci is a leading biotechnology company offering the best Bacillus licheniformis 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 licheniformis genome editing services.

Bacillus licheniformis is a Gram positive spore-forming bacterial species with highly biotechnological interests in bioflocculation, biomineralization, biofuel production, bioremediation, and anti-biofilm activity. B. licheniformis is widely used in the production of bioactive compounds and enzymes for aquaculture, agriculture, food, biomedicine, and pharmaceutical industries. Due to the development of the novel genetic manipulation tools, B. licheniformis is becoming the optimal expression vector for the production of enzymes and other bioproducts.

Our B. licheniformis 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 licheniformis Genome Editing Based on Homologous Recombination

Red/ET Recombination permits the engineering of DNA in B. licheniformis 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. licheniformis 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.

Workflow of homologous recombination genome editing

Fig.1. Workflow of homologous recombination genome editing

• Work flow:

1. Homology arms (~500 nucleotides) design and suicide plasmid construction
2. Recombineering
3. Selection/Screening
4. Validation

CRISPR /Cas9-mediated Bacillus licheniformis 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 B. licheniformis. 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.

 Workflow of CRISPR /Cas9-mediated Bacillus licheniformis Genome Editing

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

• Project steps

1. sgRNA design and construction
2. Transformation
3. Selection/Screening
4. Validation

• The advantages of CRISPR based B. licheniformis genome editing in Microbiosci:

1. Fast turnaround time
2. Scarless genome editing
3. Multigene editing: can knock-out up to 3 genes simultaneously
4. 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. licheniformis genome editing. Our talent experts are dedicated to edit your B. licheniformis 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. licheniformis genome editing service, please feel free to contact us. We are looking forward to working together with your attractive projects.

References
1. Li, Y.; Wang, H.; Zhang, L.; Ding, Z.; Xu, S.; Gu, Z.; Shi, G. Efficient Genome Editing in Bacillus licheniformis Mediated by a Conditional CRISPR/Cas9 System. Microorganisms 2020, 8, 754. https://doi.org/10.3390/microorganisms8050754
2.Cuixia Zhou, Huan Liu, Feiyan Yuan, Haonan Chai, Haikuan Wang, Fufeng Liu, Yu Li, Huitu Zhang, Fuping Lu, Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing, International Journal of Biological Macromolecules, 2019, 122, 329-337, https://doi.org/10.1016/j.ijbiomac.2018.10.170.
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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