Knockout Bacteria

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Gene-editing Microorganisms

Traditional bacterial gene editing mainly uses the RecBCD system that widely exists in bacteria to recombine the suicide plasmid with the genome to knock out the target gene. However, the efficiency of this method is extremely low, which requires repeated experiments, and may also lead to reverse mutation, and it is difficult to obtain the target gene knockout strains. λ-Red is a better choice for Escherichia coli and its close species. Its recombination efficiency will be slightly higher than the endogenous recombination system in bacteria, but the target gene needs to be replaced with a resistance gene. After successful replacement, although the recombinant enzyme can be transferred to bacteria to delete the resistance gene, it will still leave a recombination site on the genome.

Based on the above problems, the CRISPR-B™ technology system developed by Ubigene innovatively combines the Red/ET recombination system with CRISPR/Cas9 gene editing system, uses the advantages of CRISPR/Cas9 system in efficient cutting and further optimizes the gene editing plasmid and gene editing process, greatly improving the efficiency of bacterial gene editing. Compared with the previous two methods, the CRISPR-B™ technology of Ubigene can not only achieve high efficiency, but also achieve scarless knockout (that is, no residual resistant gene or recombination sites, and eliminate foreign plasmids at the same time). In addition, this technology can achieve target gene point mutation and knockin which cannot be achieved by the previous two methods. At present, Ubigene can provide gene editing (knockout/point mutation/knockin) and construction of overexpression strains of Escherichia coli, Salmonella and Pseudomonas aeruginosa, guaranteeing the delivery of positive homozygous clones.

Service Details
Escherichia coli
Escherichia coli, uses gene editing technology to modify its genome to study gene function, or change its metabolic pathway to produce a large number of commercially valuable products, so as to obtain genetically stable engineering strains that can produce specific products.
Salmonella
Salmonella is a genus of Enterobacteriaceae, gram-negative. In addition to the study of pathogens, it can also be used to establish commonly used mouse gastroenteritis models and explore more genetic tools (for example, the first extensive transduction phage P22 was found in Salmonella typhi).
Pseudomonas aeruginosa
Pseudomonas aeruginosa is a kind of conditional pathogenic bacteria that widely exists in the environment. It is also an important model organism to study the quorum sensing, biofilm, virulence and drug resistance of gram-negative bacteria.
Service type: scarless gene knockout, point mutation, knockin, gene overexpression
Deliverable: Positive clone
Turnaround:   Please inquire
Citation
Technical advantages
CRISPR-B:20X efficiency gene-editing

The efficiency is 20-30 times higher than that of the classic methods;

Scarless gene editing technology

Scarless gene-editing technology, safe and sound;

KO/PM/ KI

Easily achieve microbial gene knockout (KO), point mutation (PM) and knockin (KI);

multiple gene editing

It is possible to knockout multiple genes simultaneously.

Work Flow
CRISPR-B™ vector construction
CRISPR-B™ vector construction
CRISPR-B _CRCarrying Cas9 nuclease and Red recombinase
CRISPR-B _GCarrying donor template
CRISPR-B _DCarrying target gene gRNA
(2)Electroporation and CRISPR-B™ _CR competent cells preparation
· CRISPR-B _CR vector transfer into bacteria by electroporation. Validate the transfection by colony PCR and sequencing.
·Select the CRISPR-B _CR positive strains and prepare the CRISPR-B _CR competent cells
(3)Transfer of CRISPR-B_G and CRISPR-B _D
Transfer vectors CRISPR-B_G and CRISPR-B_D into CRISPR-B _CR competent cells by electroporation. CRISPR and Red recombination system worked together to edit the bacterial genome.
(4)Validate the single clones by PCR and sequencing. Eliminate CRISPR-B™ plasmids, and obtain the knockout clones.
Validation
1. Colony PCR     2.Sequencing

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