An important task in the post-genomic era is to study and uncover the function of genes, and the same is true for microorganisms, even with more than half of the genes in model E. coli still having unknown functions. Genetic modification of microbial genomes is a necessary step to truly harness microorganisms.
|Technical processes
CRISPR Cas9-mediated gene knock-in
1.The sgRNA sequence was designed according to the knockout gene sequence, and the Cas9-sgRNA knockout vector was constructed2.Fusion PCR amplification to obtain a recombinant repair template (missing part of the sequence of the gene of interest);3.The knockout vector and the recombinant repair template were co-transfected into the bacteria4.The Cas9-sgRNA complex cleaves the gene of interest, resulting in a double-strand break (DSB).5.The bacteria used a recombinant repair template with partial deletions of the target gene to repair the DSB to obtain the target gene knockout strain. 
RED recombination-mediated gene knock-in
1.According to the knockout gene, the homology arm sequence was designed, the knockout vector was constructed, and the knockout vector was transfected into bacteria
2.Under the action of RED recombinase, the knockout vector was homologous recombined with the target gene, and the target gene was replaced by the resistance gene, and the knockout strain was enriched by the resistance gene.
|Scope of application
|Technological advantages
1) Unique donor bacteria that do not require the target bacteria to have antibiotic resistance for insertion mutation screening.
2) Efficient donor bacterial conjugation system to ensure that the resistant plasmid has high conjugation and transfer efficiency.
3) The resistance vector is a T linearized vector, which can be directly linked to the purified PCR product without considering the enzyme cleavage site, which greatly saves the customer's time.
4) Optimized resistance vector sequences minimize redundant regions, thereby improving the efficiency of conjugation transfer and reducing the probability of non-specific recombination.
5) The resistance vector has the self-developed forward screening gene HYGRO, which greatly increases the scope of application of the resistance vector gene knockout technology and reduces the screening of recombinant mutant clones.
6) Plasmids with a series of different resistances to ensure that the target bacteria with a certain resistance can be used with suitable resistant plasmids.
|Cases1.Salmonella gene knockout and backfill experiments
2.Overexpression of E. coli genes
3.Saccharomyces cerevisiae gene knock-in
|Service** & CycleAccording to the specific situation of the strain and knockout gene evaluation**, the amount is large;The experimental period is 8-12 weeks, depending on the specificity of the strain.
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