Gene-editing Microorganisms

Home > Gene Editing Services > Microbe > Gene-editing Microorganisms

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.
Staphylococcus aureus
Staphylococcus aureus is a representative gram-positive bacteria, a common food-borne pathogenic microorganism, which can cause suppurative infection, pneumonia, enteritis, endocarditis, sepsis, and septicemia. The abuse of antibiotics can lead to the development of resistance in Staphylococcus aureus, and the effectiveness of existing treatment options is becoming increasingly unsatisfactory. Therefore, research on its drug resistance mechanism and new treatment strategies is currently of utmost importance.
Service type: scarless gene knockout, point mutation, knockin, gene overexpression
Deliverable: Positive clone
Turnaround:   Please inquire
Click to view articles citing Ubigene
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 target gene gRNA
CRISPR-B _DCarrying donor template
(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.
Gene-editing Microorganisms Work FlowGene-editing Microorganisms Work Flow
Validation
1. Colony PCR     2.Sequencing
Selected Cited Papers
E.coli with araBAD promoter knock-in - Bacterial transporter mechanism
IF=16.8
Nature Structural & Molecular Biology
E.coli with araBAD promoter knock-in - Bacterial transporter mechanism
IF=16.8 Nature Structural & Molecular Biology
Structural basis for bacterial lipoprotein relocation by the transporter LolCDE
Published by West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, Sichuan, China
Abstract:
This article used the Escherichia coli with the araBAD promoter knocked in before the lolCDE gene (constructed by CRISPR-B of Ubigene) and combined with experiments such as cryoelectron microscopy, mutagenesis, and in vivo viability testing to reveal the functional residues and structural characteristics of LolCDE, providing new insights for the sorting and transportation mechanism of outer membrane lipoproteins, and providing relevant guidance for the development of new therapies for multidrug-resistant Gram-negative bacteria.
Lipoprotein recognition in the central channel of LolCDE
Lipoprotein recognition in the central channel of LolCDE
E.coli with MutL gene knockout - Mismatch repair signal transmission mechanism
IF=16.6
Nature Communications
E.coli with MutL gene knockout - Mismatch repair signal transmission mechanism
IF=16.6 Nature Communications
MutS functions as a clamp loader by positioning MutL on the DNA during mismatch repair
Published by The Ohio State University Wexner Medical Center, USA & Chinese Academy of Sciences, China
Abstract:
In order to explore the key molecules in the signaling process of mismatch repair mechanism (MMR) and the function of PCC, the MutL gene knockout Escherichia coli constructed by Ubigene was used in this study. Through single molecule fluorescence imaging technology and in vitro reconstruction of MMR system, it was proved that MutL sliding clamp is the key molecule in MMR signaling, while MutS is only a mismatch specific clamp loader, and the function of MutL PCC was also explained. It provides new insights into the signaling mechanism of DNA mismatch repair. View details>>
MutL sliding clamp activates MMR downstream pathway
MutL sliding clamp activates MMR downstream pathway
E.coli with GspS gene knockout - Membrane translocation mechanism of secretins
IF=16.6
Nature Communications
E.coli with GspS gene knockout - Membrane translocation mechanism of secretins
IF=16.6 Nature Communications
Abstract:
The study used in situ electron cryotomography (cryo-ET) in wild-type and GspS knockout Escherichia coli (constructed by Ubigene) and reported the in situ structures of two types of T2SS secretins, the Klebsiella-type GspDα and the Vibrio-type GspDβ and revealed the translocation mechanism of these two distinct secretins, providing a new sight into the biosynthesis of secretins. View details>>
In situ structures of the GspDβ secretin on the outer and inner membranes In situ structures of the GspDβ secretin on the outer and inner membranes
In situ structures of the GspDβ secretin on the outer and inner membranes
E.coli with recA and ldhA genes knockout - Intestinal health
IF=9.2
npj Biofilms and Microbiomes
E.coli with recA and ldhA genes knockout - Intestinal health
IF=9.2 npj Biofilms and Microbiomes
Abstract:
This stuidy used pathogenic Escherichia coli ATCC 25922 as a model to targeted knockout RecA and ldhA genes (constructed by Ubigene) and found that dietary D-xylose could effectively reduce the survival rate of Escherichia coli ATCC 25922 in the intestine by activating prophage, providing a new idea for alleviating animal diarrhea and developing antibiotic substitutes. View details>>
D-xylose promotes prophage induction in E. coli ATCC 25922
D-xylose promotes prophage induction in E. coli ATCC 25922

×

Subscribe Us

By subscription, you consent to allow Ubigene Biosciences to store and process the information provided above to deliver the latest news, research spotlight, and promotions. You can unsubscribe from these communications at anytime.
×

Search documentation

Literature:

Name: *

Company: *

Telephone: *

Email:

Notes: After submitting the order, we will contact you as soon as possible.

request now