Rhizobium Gene Editing Service
Rhizobium Gene Editing Service
As a key microorganism symbiotically fixing nitrogen with leguminous plants, rhizobium’s nitrogen fixation efficiency, environmental adaptability, and host specificity directly determine the nitrogen cycle efficiency of agricultural ecosystems and the yield of leguminous crops. The application of precise gene editing technologies provides core technical support for breaking the inherent limitations of natural rhizobium, developing high-efficiency nitrogen-fixing engineering strains, and promoting the development of green agriculture. Relying on the microbial genome modification technology coordinated by the CRISPR/Cas9 and Tn5 transposase systems, we can provide customized services for rhizobium, including marker-free gene knockout, scarless point mutation, gene knock-in/overexpression, and large-fragment deletion. We control the experimental process throughout, deliver high-purity positive engineering strains, and assist in the implementation of projects related to agricultural research, microbial research, and green agricultural industries.
I. Strain Characteristics and Biological Background
1.1 Core Biological Characteristics
Rhizobium is a Gram-negative bacillus belonging to the phylum Proteobacteria, widely distributed in soil environments. Its core characteristic is the ability to form symbionts—nodules—with leguminous plants (such as soybeans, peanuts, and milk vetch). Through nitrogenase, it converts free nitrogen in the atmosphere into ammonia nitrogen that plants can directly absorb and utilize, without relying on chemical nitrogen fertilizers. It is a core participant in the natural nitrogen cycle and the most valuable biological nitrogen fixation resource in agricultural production.
1.2 Research and Application Value
Natural rhizobium generally has problems such as low nitrogen fixation efficiency, narrow environmental adaptability (weak tolerance to acid-base, high temperature, drought, and soil heavy metals), and single host range, which limit its field application effect. As a model strain for studying microbe-plant symbiotic interaction, the molecular mechanism of biological nitrogen fixation, and the engineering modification of agricultural microorganisms, the gene editing and modification of rhizobium not only has important theoretical research significance but also provides a practical technical path for the development of green agriculture.
1.3 Industrial and Ecological Significance
Optimizing rhizobium traits through gene editing can significantly improve its symbiotic nitrogen fixation capacity, reduce the total application of chemical nitrogen fertilizers, reduce agricultural non-point source pollution, and improve the soil ecological environment. At the same time, the modified engineering strains can expand the host range, enhance stress resistance, adapt to soil conditions in different regions, promote the development of agriculture towards green, ecological, and sustainable directions, and have significant industrial application value and ecological benefits.
II. Literature-Reported Editing Strategies
Combined with the genomic characteristics of rhizobium (Gram-negative bacteria with complex cell membrane structure and high transformation difficulty), the mainstream editing strategies adopted in current scientific research and practice take the CRISPR/Cas9 system as the core, combined with Tn5 transposase, λRed/ET homologous recombination and other technologies, forming an efficient, precise, and convenient modification system, as follows:
2.1 CRISPR/Cas9 System (Core Editing Scheme)
Principle: Using Cas9 nuclease to precisely recognize and double-strand cleave the target site in the rhizobium genome under the specific guidance of sgRNA (single-guide RNA), and then realize precise knockout, scarless point mutation, or exogenous gene knock-in of the target gene with the help of the host’s own homology-directed repair (HDR) mechanism.
Advantages: Extremely strong targeting and extremely low off-target rate, with editing efficiency significantly higher than traditional recombination technologies; combined with the scarless editing process, it can completely eliminate exogenous resistance markers and plasmid residues, obtain homozygous and stable engineering strains, which meet biosafety requirements.
2.2 Tn5 Transposase System (Auxiliary for Efficient Integration)
Principle: Using Tn5 transposase to recognize specific transposon sequences, mediate the random or directed insertion of transposons (carrying target genes or marker genes) in the rhizobium genome, quickly realize the integration and stable expression of exogenous genes, without relying on homologous arm design.
Advantages: Simple operation and high integration efficiency; no complex vector construction or homologous arm design is required, which can quickly realize target gene overexpression or strain fluorescent labeling; when used in coordination with the CRISPR/Cas9 system, it can greatly improve editing efficiency and success rate.
2.3 Electroporation Optimization (Key Auxiliary Technology)
Aiming at the problems of complex cell membrane structure and high difficulty in plasmid introduction of rhizobium, optimize electroporation parameters (voltage, pulse time, bacterial concentration, etc.), prepare high-competent rhizobium cells, efficiently introduce editing vectors (CRISPR plasmids, Tn5 transposons) into the bacteria, break the transformation barrier of Gram-negative bacteria, and improve transformation efficiency and editing success rate.
Figure 1: Schematic Diagram of CRISPR/Cas9 Cleavage and Tn5 Transposition
III. Core Application Fields
Centered on "improving nitrogen fixation efficiency, enhancing environmental adaptability, and expanding application scenarios", rhizobium gene editing services are widely used in agricultural research, microbial research, and green agricultural industries. The specific application directions are as follows:
3.1 Optimization of Biological Nitrogen Fixation Efficiency
By knocking out nitrogen fixation-related negative regulatory genes (such as nifL gene), enhancing the expression level of nitrogenase activity-related genes (such as nifH and nifD genes), optimizing the nitrogen fixation metabolic pathway of rhizobium, improving its symbiotic nitrogen fixation capacity, providing sufficient nitrogen nutrition for leguminous crops, and reducing dependence on chemical nitrogen fertilizers.
3.2 Modification of Environmental Adaptability
Edit stress resistance-related genes (such as acid-base tolerance genes, drought resistance genes, and heavy metal resistance genes) to improve the survival, colonization, and nitrogen fixation capacity of rhizobium in extreme soil environments (acidic, alkaline, arid, and heavy metal-contaminated soils), expand its field application range, and adapt to agricultural production conditions in different regions.
3.3 Expansion of Host Range
Break the host specificity limitation of natural rhizobium through genetic modification, edit host recognition-related genes (such as nodD gene), enable it to symbiose, nodulate, and fix nitrogen with more types of leguminous plants (even non-leguminous plants), improve the nitrogen cycle utilization efficiency of the ecosystem, and expand application scenarios.
3.4 Research on Microbe-Plant Interaction
Insert reporter genes (such as green fluorescent protein GFP and red fluorescent protein mScarlet3) into the rhizobium genome to construct fluorescently labeled strains, real-time monitor the colonization, infection, nodulation, and nitrogen fixation dynamics of rhizobium in plant roots, and provide tool strains for analyzing the molecular mechanism of microbe-plant symbiotic interaction.
3.5 Construction of High-Quality Engineering Strains
Adopt multi-gene continuous editing or multi-site simultaneous editing technology to construct high-quality rhizobium engineering strains with high nitrogen fixation efficiency, strong stress resistance, and wide host range, which are applied in green agricultural production to achieve the goal of "reducing nitrogen and increasing efficiency, and ecological environmental protection".
IV. Project Process and Verification Standards
We provide one-stop rhizobium gene editing services from scheme design to strain delivery, strictly follow standardized experimental processes, and conduct strict quality control at each step to ensure the accuracy, stability, and reliability of editing results. The specific process is as follows:
4.1 Scheme Design and Vector Construction
Combine customer needs (gene knockout/knock-in/point mutation/overexpression, etc.), analyze the target gene sequence of rhizobium, and design an exclusive editing scheme; construct CRISPR editing plasmids (containing sgRNA and Cas9 gene) or Tn5 transposon vectors (containing target genes and marker genes), and verify the correctness of vector construction by sequencing.
4.2 Bacterial Transformation and Candidate Strain Screening
Prepare high-competent rhizobium cells, introduce editing vectors into the bacteria by electroporation; after culture, obtain preliminary candidate positive strains through resistance screening (such as antibiotic screening) or phenotypic screening, and eliminate untransformed wild-type strains.
4.3 Multiple Verification (Core Quality Control Link)
1. PCR Identification: Design specific primers for the target site, perform PCR amplification on candidate strains to verify whether the target gene is successfully knocked out, knocked in, or mutated;
2. DNA Sequencing Verification: Sequence the target region of PCR-positive strains to confirm the accuracy of the editing site, no off-target effect, and the edited sequence meets expectations;
3. Functional Verification: For overexpression projects, additionally detect the mRNA transcription level and protein expression level of the target gene; for nitrogen fixation-related modification projects, detect the nitrogenase activity of the strain to ensure that trait optimization meets the standard;
4. Homozygosity Verification: Detect the stability of the edited traits of the strain through continuous subculture to ensure the acquisition of homozygous positive engineering strains.
Figure 2: Schematic Diagram of the Project Process
V. Details of Gene Editing Projects
5.1 Core Service Content
Relying on a mature editing technology system, we can provide comprehensive rhizobium gene editing services to meet different research and application needs. The specific services include:
• Marker-free Gene Knockout: Precisely knock out target genes, completely eliminate exogenous plasmids and resistance markers without any residual sequences, suitable for gene function research and knocking out negative regulatory genes to optimize traits such as nitrogen fixation;
• Scarless Gene Point Mutation: Precisely introduce single or multiple base mutations at the target site of the rhizobium genome without changing other sequences, used for studying gene function, modifying protein structure (such as nitrogenase structure), and improving enzyme activity or stress resistance;
• Scarless Gene Knock-in: Precisely insert exogenous functional genes (such as nitrogen fixation-enhancing genes) or reporter genes (such as fluorescent protein genes) at specific sites in the genome to achieve strain trait upgrading or labeling without residual exogenous markers;
• Gene Overexpression: Realize site-specific or random overexpression of target genes through the Tn5 transposase system or CRISPR/Cas9 system, enhance the expression level of nitrogen fixation, stress resistance and other related genes, and quickly improve the target traits of the strain;
5.2 Core Technical Advantages
• High Editing Efficiency: The CRISPR/Cas9 system cooperates with λRed homologous recombination technology, the single-site gene editing efficiency can reach more than 90%, and the large-fragment modification efficiency can reach more than 75%, which greatly reduces experimental costs and cycles;
• Rapid and Efficient Integration: The Tn5 transposase system does not require homologous arm design, can quickly realize exogenous gene integration and overexpression, and shorten the construction cycle of engineering strains by more than 30%;
• Scarless and Precise Editing: No exogenous markers, plasmid residues, or recombination sites are left at the edited target site, which meets biosafety requirements, and the strain has high homozygosity and stable traits;
• Simple and Controllable Operation: Standardized experimental process, simplified editing plasmid construction and electroporation operation, reducing the threshold of experimental technology; at the same time, provide experimental data and quality control reports throughout the process to ensure traceable processes;
• Strong Targeting and Low Off-Target Rate: The CRISPR/Cas9 system precisely recognizes target sequences through sgRNA, with an off-target rate of less than 1%, avoiding non-specific genome modification and ensuring the reliability of editing results.
VI. Introduction to Typical Cases
We have successfully provided rhizobium gene editing services for many agricultural research institutions and microbial research laboratories, accumulated rich experimental experience, and can efficiently meet the editing needs of different rhizobium strains (Bradyrhizobium, Rhizobium, etc.). The following is a typical case:
Case: Fluorescent Labeling Modification of Bradyrhizobium Based on the Tn5 Transposase System
Project Requirements
The customer needed to construct a red fluorescently labeled Bradyrhizobium engineering strain for subsequent research on microbe-soybean symbiotic interaction, requiring stable fluorescent signals, no obvious growth inhibition, and realizable in vivo monitoring.
Technical Route
1. Construction of Fusion Vector: Fusion the red fluorescent protein gene mScarlet3 with a resistance gene (kanamycin resistance), insert it into the Tn5 transposon vector, and verify the correctness of the vector by sequencing;
2. Preparation of Transposome Complex: Incubate Tn5 transposase with the constructed transposon vector to form Tn5 Transposome RNP Complex;
3. Electroporation and Screening: Introduce the transposome complex into Bradyrhizobium competent cells by electroporation, spread on medium containing kanamycin, and screen for resistance-positive strains;
4. Verification and Screening: Observe the expression of fluorescent protein in positive strains by fluorescence microscope; verify the stability of transposon integration by PCR and sequencing; conduct continuous subculture to confirm that the fluorescent signal does not attenuate.
Experimental Results
Three Bradyrhizobium engineering strains stably expressing red fluorescence were successfully obtained. The fluorescent signals were clear and uniform, and the growth rate of the strains was not significantly different from that of the wild type, without growth inhibition; the fluorescent signal could be stably passaged for more than 10 generations, which could be directly used for in vivo monitoring research on soybean root colonization and nodulation dynamics, fully meeting the customer’s experimental needs.
Figure 3: Fluorescent Labeling Results (Escherichia coli and Bradyrhizobium)
VII. References
[1] Oldroyd, G.E.D. & Dixon, R. Biotechnological solutions to the nitrogen problem. Current Opinion in Biotechnology 26, 19–24 (2014). DOI: 10.1016/j.copbio.2013.08.006.
[2] Wang, L. et al. Genetic engineering of nitrogen-fixing bacteria: recent advances and future prospects. Biotechnology Advances 40, 107546 (2020). DOI: 10.1016/j.biotechadv.2019.107546.
[3] Mus, F. et al. Symbiotic nitrogen fixation and the challenges to its extension to nonlegumes. Applied and Environmental Microbiology 82, 3698–3710 (2016). DOI: 10.1128/AEM.01055-16.
[4] Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products (λ-Red recombineering). PNAS 97, 6640–6645 (2000). DOI: 10.1073/pnas.120163297.
[5] Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31, 233–239 (2013). DOI: 10.1038/nbt.2508.
[6] Zhang, Y. et al. CRISPR/Cas9-mediated gene editing in Rhizobium fredii improves symbiotic nitrogen fixation with soybean. Journal of Biotechnology 345, 113987 (2022). DOI: 10.1016/j.jbiotec.2022.113987.
Cooperate with Us
We have an experienced and technically solid microbial gene editing team. Relying on the mature CRISPR/Cas9 and Tn5 transposase coordinated editing technology, we are deeply engaged in the field of rhizobium genetic modification. We can tailor exclusive editing schemes according to customers’ specific needs (different strains, different editing targets, different application scenarios).
We promise: With professional technical support, rigorous experimental processes, and efficient communication services, we will control the experimental quality throughout, ensure the project is delivered on time and the results meet the standards, and help your agricultural microbial research, engineering strain development and other projects advance rapidly.
Consult now to get your exclusive rhizobium gene editing scheme and detailed quotation!
