Bacillus thuringiensis (or Bt) is a Gram-positive, soil-dwelling bacterium, the most commonly used biological pesticide worldwide. B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well on leaf surfaces, aquatic environments, animal feces, insect-rich environments, and flour mills and grain-storage facilities. It has also been observed to parasitize other moths such as Cadra calidella.
Figure 1 Gram stain of Bacillus thuringiensis under magnification
Our B. thuringiensis genome editing services based on CRISPR/Cas9 technology and homologous recombination technique. The B. thuringiensis genome editing system helps you successfully achieve gene knockout, gene insertion and point mutation for either research or industrial purposes. The Bt genes engineered into crops and approved for release include, singly and stacked: Cry1A.105, CryIAb, CryIF, Cry2Ab, Cry3Bb1, Cry34Ab1, Cry35Ab1, mCry3A, and VIP, and the engineered crops include corn and cotton.
Red/ET Recombination permits the engineering of DNA in B. thuringiensis 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. thuringiensis 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 thuringiensis. 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|
|• Homology arms design and suicide plasmid construction||• sgRNA design and construction|
|• Recombineering||• Transformation|
|• Selection/Screening||• Selection/Screening|
|• Validation||• Validation|
Creative Bioene offers the best Bacillus thuringiensis 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 thuringiensis genome editing services. If you have any special requirements in our B. thuringiensis genome editing service, please feel free to contact us. We are looking forward to working together with your attractive projects.