Bacterial Genome Editing

Bacteria is the most abundant type of organisms in nature. The modification of the bacterial genome helps us understand the pathogenic mechanism of pathogenic bacteria, the mechanism of drug resistance, and eliminate drug-resistant bacteria. It can also improve the fermentation of industrial strains, synthesize new secondary metabolites, and enrich biological raw materials.

Creative Biogene has many years of experience in bacterial mutant library construction, homologous recombination and CRISPR genetic modification.

Examples of bacteria with successful gene editing

CRISPR /Cas9-mediated Bacteria Genome Editing

CRISPR-Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. Three types (I–III) of CRISPR systems have been identified across a wide range of bacterial and archaeal hosts, wherein each system comprises a cluster of CRISPR-associated (Cas) genes, noncoding RNAs and a distinctive array of repetitive elements (direct repeats). . These repeats are interspaced by short variable sequences derived from exogenous DNA targets known as protospacers, and together they constitute the CRISPR RNA (crRNA) array. Within the DNA target, each protospacer is always associated with a protospacer adjacent motif (PAM), which can vary depending on the specific CRISPR system.

Project Flow

According to the different characteristics of each bacteria, a CRISPR/Cas9 system can be developed.

1) Project evaluation and design of CRISPR/Cas9 editing experiments.
2) Cultivate the strains provided by clients and perform 16s sequencing to verify the species.
3) Conduct drug sensitivity screening assays and determine the marker gene.
4) Use online software tools to design gRNA.
5) Design homology arms for the repair process.
6) Construct resistance marker gene, gRNA and homology arms into CRISPR/Cas9 plasmid.
7) Prepare the competent strain.
8) High-efficiency transformations.
9) Screen the successfully edited strains following with sequencing and identification.

Provided by Clients

The name of the bacteria.
The requirements of culture conditions.
The results of the whole genome sequencing.
The target gene information.

Deliverables

√ Plasmid map for editing
√ The successfully edited strain
√ Experiment reports (experimental procedures, sequencing files, related pictures, etc.)

Homologous Recombination-mediated Genome Editing

Homologous recombination, the exchange of genetic material between two strands of DNA that contain long stretches of similar base sequences. Homologous recombination occurs naturally in eukaryotic organisms, bacteria, and certain viruses and is a powerful tool in genetic engineering.

Gene modification methods applied in bacteria

Method based on Red/ET system
Method based on suicide vectors
Method based on temperature-sensitive plasmids

Creative Biogene combines different homologous recombination methods according to the characteristics of different bacteria to meet the requirements of genome modification, such as knockout, replacement, insertion or site-directed mutation.

Project Flow

According to the different characteristics of each bacteria, a homologous recombination system can be developed.

1) Project evaluation and design of homologous recombination system.
2) Cultivate the strains provided by clients and perform 16s sequencing to verify the species.
3) Conduct drug sensitivity screening assays and determine the marker gene.
4) Design the homology arms flanking each side of the gene.
5) Construct resistance marker gene and homology arms into the plasmid used in homologous recombination system.
6) Prepare the competent strain.
7) High-efficiency transformations.
8) Screen the successfully edited strains following with sequencing and identification.

Provided by Clients

The name of the bacteria.
The requirements of culture conditions.
The whole genome sequencing result or database link.
The target gene information along with its upstream and downstream sequences.

Deliverables

√ Plasmid map for homologous recombination editing
√ The successfully edited strain
√ Experiment reports (experimental procedures, sequencing files, related pictures, etc.)

We have established a mature microbial genome editing platform to satisfy your research requirements. If you are interested in our bacterial genome editing services, please contact us for more details.