Knockout Bacteria

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Knockout Bacteria

·    Gene-editing microorganisms    ·
Red/ET recombination system is a classical method of microbial gene editing, which can achieve the knockin, knockout, point mutation and other modifications of the target gene. This technology has been widely used in genetic modification of genomic DNA, such as bacterial artificial chromosome(BAC), Escherichia coli chromosome. However, the efficiency of this system still needs to be improved. How to improve the efficiency of gene recombination and editing has always been a hotspot of microbial gene editing. Therefore, CRISPR/Cas9 technology is adopted to improve the efficiency of microbial gene editing.

CRISPR/Cas9 is an acquired immune system in bacteria and archaea and can be used to fight against invading viruses and exogenous DNA. In recent years, the CRISPR/Cas9 gene-editing technology has been widely used because it is simple and efficient. It has been the most advanced method for gene editing.

Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi.
·  Key Features  ·
The efficiency is 20-30 times higher than that of the classic methods;
Scarless gene-editing technology, safe and sound;
Easily achieve microbial gene knockout (KO), point mutation (PM) and knockin (KI);
It is possible to knockout multiple genes simultaneously.
·   Editing bacteria  ·
Ubigene uses CRISPR-B™ system to modify the genome of fungi and achieve knockout, point mutation or knockin. Customers could choose the genome editing methods with or without residual.
What bacteria can CRISPR-B™ modify?
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Some gram-positive bacteria

Bacillus subtilis

Streptococcus thermophilus

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Most of the gram-negative bacteria

Escherichia coli

Salmonella

Pseudomonas aeruginosa

For more bacteria species, please consult us.
· Work flow  ·
CRISPR-B™ vector construction
CRISPR-B™
vector construction
CRISPR-B _CR Carrying Cas9 nuclease and Red recombinase

CRISPR-B _G Carrying donor template
CRISPR-B _D Carrying target gene gRNA
Electroporation and CRISPR-B™ _CR competent cells preparation
1. CRISPR-B _CR vector transfer into bacteria by electroporation. Validate the transfection by colony PCR and sequencing.
2. Select the CRISPR-B _CR positive strains and prepare the CRISPR-B _CR competent cells
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.
Validate the single clones by PCR and sequencing. Eliminate CRISPR-B™ plasmids, and obtain the knockout clones.
技术流程
Validation
1. Colony PCR     2.Sequencing