Bacillus licheniformis is a bacterium commonly found in the soil. It is a gram-positive, mesophilic bacterium. Its optimal growth temperature is around 50 °C, though it can survive at much higher temperatures. The optimal temperature for enzyme secretion is 37 °C. It can exist in a dormant spore form to resist harsh environments, or in a vegetative state when conditions are good. High capacity of secretion of the alkaline serine protease has made B. licheniformis one of the most important bacteria in industrial enzyme production. Subtilisin Carlsberg, a serine protease, secreted by B. licheniformis is used as a detergent protease. A small antisense RNA against Subtilisin Carlsberg named BLi_r0872 was discovered in an RNA-seq based study. It may have a putative impact on protease production and serve as target for strain improvement. Amylases are also synthesized by B. licheniformis and used for industrial purposes. B. licheniformis is used as a probiotic in animal feed, where isolates have been shown to prevent disease and promote growth. B. licheniformis also shows possible applications in bioremediation, biomineralization, and biofuels as more examples.
Figure 1 Bacillus licheniformis colonies on a blood agar plate.
Our B. licheniformis genome editing services are based on CRISPR/Cas9 technology and homologous recombination technique. The B. licheniformis genome editing system helps you successfully achieve gene knockout, gene insertion and point mutation for either research or industrial purposes.
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.
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 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.
|Homologous Recombination System
|CRISPR /Cas9 System
Bacillus licheniformis is widely used to produce multiple enzymes and chemicals in industrial fermentation. It is also an organism that is hard to genetically manipulate, which is mainly attributed to its extremely low transformation efficiency. The lack of genetic modification technology severely limits its further application. Researchers (Youran Li, et al. 2020) has developed an all-in-one conditional clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 plasmid for B. licheniformis with the cas9 gene under the control of a xylose-inducible promoter. It suggests that the design with a cas9 gene under the strict control of a xylose-inducible promoter significantly improved the success rate of genome editing in this host. This work contributes to the development of genetic manipulation and furthers the use of B. licheniformis as an efficient industrial workhorse.
Creative Biogene offers 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. 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.