Authors
Creative Biogene
Summary
Creative Biogene 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.
Introduction
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.
Materials
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.
Advertisement
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 332
- Comments 0