Escherichia coli

Genome Editing Service for Escherichia coli

1. Research Background

Escherichia coli is the most classic model organism in life sciences research and a core chassis cell in synthetic biology and the biotechnology industry. Although its genetic background is well-defined, precise and efficient gene-editing tools are still required for complex metabolic pathway engineering, multi-gene sequential editing, and work with specific clinical isolates (such as hypervirulent or multidrug-resistant strains). Based on mature CRISPR/Cas9 and λ-Red recombination systems, GeneRulor provides professional, customized E. coli genome editing solutions for global clients.

2. Strain Characteristics and Biological Background

(1) Gram-staining property:   Escherichia coli is a Gram-negative bacterium. Its physical characteristic is a relatively thin peptidoglycan layer, which is externally enveloped by a complex outer membrane structure (containing lipopolysaccharide, LPS), distinguishing it significantly from Gram-positive bacteria.

(2) Clinical and Research Significance: It is both part of the normal gut microbiota and includes pathogenic strains (e.g., EHEC, ETEC). In the research field, it is the model organism of choice for cloning and expression, metabolic engineering, and fundamental genetics studies.

(3) Metabolism and Applications: Possessing strong environmental adaptability and rapid growth capability, it serves as a crucial platform for biofilm research, heterologous protein expression, and the industrial production of bio-based small molecules.

Figure 1. Growth of E. coli on LB Agar Plates

3. Gene-Editing Strategies Reported in the Literature

Given the genetic characteristics of E. coli, mainstream editing strategies in current literature and research practice include:

3.1 CRISPR/Cas9 System (Primary Solution):

(1) Principle: Utilizes the Cas9 protein to perform precise cleavage of target genomic DNA under the guidance of sgRNA, inducing double-strand breaks (DSBs).

(2) Advantage: When combined with donor DNA, it enables scarless knockout, point mutations, and large-fragment integration, with screening efficiency far surpassing traditional methods.

3.2 λ-Red Homologous Recombination System:

A classic strategy unique to E. coli, it utilizes the three proteins Gam, Exo, and Beta to mediate recombination between linear DNA fragments and the genome, suitable for rapid gene inactivation.

3.3 Site-Specific Recombination (e.g., Cre/loxP):

Used for the excision of antibiotic resistance markers, enabling multiple rounds of sequential editing.

3.4 Transformation Efficiency Optimization:

For clinical strains that are difficult to transform, optimization of electroporation parameters or the use of chemical competence preparation techniques helps overcome the outer membrane barrier.

4. Core Application Areas

(1) Metabolic Engineering: Precise knockout of genes in byproduct metabolic pathways to optimize precursor accumulation and enhance target product yield.

(2) Synthetic Biology: Insertion of large biosynthetic gene clusters or reporter genes (e.g., GFP, mCherry) at specific sites (e.g., attB sites) to construct intelligent biosensors.

(3) Drug Resistance and Virulence Studies: Simulating the evolution of resistance genes through precise point mutations to study the interaction between E. coli and the host immune system.

(4) Engineered Strain Development: Sequential editing of multiple genes to construct specialized expression hosts with deleted endotoxins or reduced protease activity.

5. Project Workflow and Validation

We provide a one-stop service from strategy design to mutant strain delivery:

(1) Strategy Design and Vector Construction: Designing optimal sgRNAs and homologous arms for the target sites.

(2) Bacterial Transformation and Recombination: Introducing the editing system into E. coli via electroporation.

(3) Screening and Plasmid Curing: Utilizing antibiotic selection and methods like temperature induction to eliminate editing plasmids.

(4) Multi-level Validation: Providing reports on PCR identification, Sanger sequencing, and phenotypic analysis.

Figure 2. Schematic Diagram of Project Workflow

6. Genome Editing Project Introduction

6.1 Core Services Include:

(1) Gene Knockout/Inactivation: Achieving complete deletion or frameshift mutations of target genes.

(2) Gene Knock-in/Integration: Insertion of exogenous sequences at safe genomic loci to construct stable expression strains.

(3) Precise Point Mutation: Achieving precise substitution of single or multiple nucleotides.

(4) Multi-gene Sequential Editing: Realizing multi-target modifications in a single experiment or multiple rounds based on CRISPR technology.

6.2 Technical Advantages:

(1) Extensive Experience: Coverage includes strains like MG1655, 1917, DH5α, and various clinical isolates.

(2) Scarless Editing: No residual antibiotic resistance markers, meeting the requirements for subsequent experiments and industrial applications.

(3) Fast Delivery Cycle: Optimized processes ensure the delivery of sequencing-verified positive strains in the shortest possible time.

7. Case Studies

We have successfully provided services for numerous top universities, research institutes, and biotech companies both domestically and internationally. Below is an example:

Case: Genome Modification of Escherichia coli MG1655

Project Description: Successfully knocked out the target gene in the Escherichia coli MG1655 genome.

Figure 3. Monoclonal Identification of Target Gene Knockout

Figure 4. Sequencing Results Confirming Successful Target Gene Knockout

8. References

[1] Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. PNAS.

[2] Jiang, Y., et al. (2015). Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Applied and Environmental Microbiology.

[3] Li, Y., et al. (2015). Metabolic engineering of Escherichia coli using CRISPR-Cas9 system. Metabolic Engineering.

Cooperate with Us

By choosing us, you will gain an experienced and technically proficient partner in genome editing. We commit to accelerating your research or projects with professional technologies, rigorous processes and efficient communication.

Consult us now to obtain your customized editing scheme and quotation!