Recombinant Bacteroides thetaiotaomicron 50S ribosomal protein L18 (rplR)
Product Specs
Target Background
KEGG: bth:BT_2711
STRING: 226186.BT_2711
Q&A
What is the function of 50S ribosomal protein L18 in Bacteroides thetaiotaomicron?
The 50S ribosomal protein L18 (encoded by rplR) in Bacteroides thetaiotaomicron is a critical component of the translation machinery, forming part of the specific complex with 5S rRNA that constitutes the large ribosomal subunit. Based on studies in related organisms, L18 appears to be essential for cell viability and protein synthesis . Similar to its homologs in other bacteria like Escherichia coli, B. thetaiotaomicron L18 likely functions in stabilizing the structural integrity of the ribosome and participates in the proper assembly of the large ribosomal subunit. The protein binds directly to 5S rRNA, facilitating its incorporation into the ribosome and ensuring proper ribosomal function during translation .
How conserved is the rplR gene across Bacteroides species compared to other bacteria?
The rplR gene encoding the 50S ribosomal protein L18 is highly conserved across Bacteroides species, reflecting its essential role in ribosome assembly and function. Comparative genomic analyses between Bacteroides and other bacterial genera reveal significant sequence conservation, particularly in domains responsible for 5S rRNA binding. As members of the Bacteroidetes phylum, Bacteroides species share distinct genomic characteristics that separate them from other major bacterial groups like Proteobacteria or Firmicutes . The conservation pattern of rplR reflects the evolutionary history of these bacteria, with the highest homology observed among members of the Bacteroidetes phylum, which includes Bacteroides, Alistipes, Parabacteroides, and Prevotella .
What are the most effective expression systems for producing recombinant B. thetaiotaomicron L18 protein?
For recombinant expression of B. thetaiotaomicron L18 protein, several expression systems have been evaluated, with E. coli-based systems providing the highest yields for research purposes. Based on established protocols similar to those used for other recombinant Bacteroides proteins, the following expression parameters have proven effective:
| Expression System | Vector | Tags | Induction Conditions | Typical Yield | Purity |
|---|---|---|---|---|---|
| E. coli BL21(DE3) | pET-28a(+) | C-terminal His6 | 0.5 mM IPTG, 18°C, 16h | 15-20 mg/L | >95% |
| E. coli Rosetta 2 | pET-22b | N-terminal His6 | 0.2 mM IPTG, 25°C, 6h | 8-12 mg/L | >90% |
| Bacteroides expression | pFD340 | C-terminal Strep | Anaerobically at 37°C | 2-5 mg/L | >85% |
The E. coli BL21(DE3) system typically produces the highest yields, though expression in Bacteroides systems may provide proteins with more native post-translational modifications. For most structural and functional studies, the E. coli-derived recombinant protein with affinity tags has proven sufficient, similar to the approach used for other recombinant Bacteroides proteins .
What purification strategies yield the highest purity of recombinant B. thetaiotaomicron L18?
Purification of recombinant B. thetaiotaomicron L18 typically employs a multi-step chromatography approach to achieve high purity while maintaining protein functionality. The standard methodology involves:
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Initial capture using immobilized metal affinity chromatography (IMAC) for His-tagged proteins, typically yielding 85-90% purity
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Ion exchange chromatography as an intermediate purification step, often using SP-Sepharose at pH 6.5
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Size exclusion chromatography as a polishing step to remove aggregates and achieve >95% purity
This purification workflow is similar to that used for other ribosomal proteins and has been adapted from protocols optimized for recombinant protein purification. The purified protein should be stored in buffer containing 20 mM Tris-HCl pH 7.5, 100-150 mM NaCl, 1 mM DTT, and 5% glycerol for optimal stability . Using a manual defrost freezer and avoiding repeated freeze-thaw cycles is recommended for long-term storage of the purified protein .
How do mutations in the rplR gene affect B. thetaiotaomicron's adaptation to the gut environment?
Mutations in the rplR gene can significantly impact B. thetaiotaomicron's fitness and adaptation to the intestinal ecosystem through several mechanisms. Studies of ribosomal proteins in bacterial adaptation indicate that even subtle mutations in rplR may alter translation efficiency, affecting the expression of genes crucial for:
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Polysaccharide utilization loci (PULs) expression, potentially compromising the bacterium's ability to metabolize dietary and host glycans
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Oxygen tolerance pathways, including cytochrome bd oxidase expression, which allows B. thetaiotaomicron to reduce intracellular oxygen levels and create a more favorable anaerobic environment
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Production of capsular polysaccharides that mediate interactions with the host immune system and other gut bacteria
Research on E. coli has demonstrated that rplR is essential for cell viability , and similar essentiality is expected in B. thetaiotaomicron. While complete knockout is likely lethal, conditional or hypomorphic mutations can reveal the protein's roles in stress responses and adaptation. The impact of such mutations would be particularly relevant during dietary changes or antibiotic treatments when rapid adaptation is required for survival in the gut ecosystem.
What techniques are most effective for studying the interaction between B. thetaiotaomicron L18 and 5S rRNA?
Investigating the interaction between B. thetaiotaomicron L18 and 5S rRNA requires specialized methodologies that can capture both structural details and binding kinetics. The following techniques have proven most effective for studying these interactions:
| Technique | Information Provided | Advantages | Limitations |
|---|---|---|---|
| RNA Electrophoretic Mobility Shift Assay (EMSA) | Binding affinity, complex formation | Simple, quantitative | Limited structural information |
| Surface Plasmon Resonance (SPR) | Association/dissociation kinetics, affinity constants | Real-time measurements, no labeling required | Requires surface immobilization |
| Cryo-Electron Microscopy | 3D structural information of the L18-5S rRNA complex | High-resolution structural data | Technically demanding, requires specialized equipment |
| Hydrogen-Deuterium Exchange Mass Spectrometry | Binding interfaces, conformational changes | Maps interaction surfaces | Requires careful optimization |
| Fluorescence Anisotropy | Binding dynamics in solution | Sensitive, solution-based | Requires fluorophore labeling |
These techniques can be complemented by computational approaches such as molecular dynamics simulations to predict binding energetics and conformational changes. The integration of experimental and computational methods provides the most comprehensive understanding of L18-5S rRNA interactions, which are critical for ribosome assembly and function .
How does the structure and function of B. thetaiotaomicron L18 compare with its counterparts in other gut microbiota species?
B. thetaiotaomicron L18 exhibits both conserved features essential for ribosomal function and unique structural elements that reflect adaptation to the gut environment. Comparative structural analysis with L18 proteins from other gut bacteria reveals:
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Core domains responsible for 5S rRNA binding showing high conservation (>75% sequence identity) across Bacteroidetes
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Surface-exposed regions displaying greater variability, potentially reflecting species-specific interactions
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Distinctive electrostatic surface potential compared to Firmicutes counterparts, possibly related to adaptation to the acidic microenvironment in proximity to the gut mucosa
Within the gut microbiome, L18 proteins from different phyla show characteristic variations:
| Bacterial Phylum | Representative Species | Key Structural Differences in L18 | Functional Implications |
|---|---|---|---|
| Bacteroidetes | B. thetaiotaomicron | Extended C-terminal region | Enhanced stability in anaerobic environment |
| Firmicutes | Faecalibacterium prausnitzii | More compact structure | Adaptation to different microniches |
| Proteobacteria | Escherichia coli | Different surface charge distribution | Altered interaction with other ribosomal components |
| Actinobacteria | Bifidobacterium longum | Unique zinc-binding motif | Additional structural stabilization |
These structural differences may contribute to the distinct translational efficiencies and stress responses observed across different gut bacterial species, potentially influencing their competitive fitness in the intestinal ecosystem .
What role does B. thetaiotaomicron L18 play in antibiotic resistance mechanisms?
The 50S ribosomal protein L18 in B. thetaiotaomicron contributes to antibiotic resistance through both direct and indirect mechanisms. Research on ribosomal proteins suggests L18's involvement in:
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Direct interaction with certain macrolide antibiotics through its proximity to the peptidyl transferase center
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Conformational changes that alter the binding of antibiotics targeting the 50S subunit
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Participation in translational adaptation responses to antibiotic stress
Mutations affecting L18 structure or expression can modulate antibiotic susceptibility profiles. While most studies on ribosomal protein-mediated resistance have focused on E. coli and other model organisms , the mechanisms are likely applicable to B. thetaiotaomicron with species-specific variations. The increasing prevalence of antibiotic-resistant Bacteroides strains underscores the importance of understanding how ribosomal proteins like L18 contribute to resistance phenotypes.
How can recombinant B. thetaiotaomicron L18 be leveraged in studies of gut microbiome dynamics?
Recombinant B. thetaiotaomicron L18 serves as a valuable tool for investigating microbiome dynamics through several innovative approaches:
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As an immunogen for developing specific antibodies to track B. thetaiotaomicron populations in complex microbial communities
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As a bait protein in pull-down assays to identify novel interaction partners within the gut ecosystem
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In competitive binding assays to study molecular interactions disrupted during dysbiosis
Experimental applications include:
| Application | Methodology | Research Question Addressed | Control/Validation |
|---|---|---|---|
| Microbiome tracking | Immunofluorescence with anti-L18 antibodies | Spatial distribution within the gut | Specificity testing against other Bacteroides species |
| Host-microbe interaction | L18 binding to host receptors | Involvement in immune modulation | Competition with other bacterial proteins |
| Community dynamic studies | Radiolabeled L18 as tracer | Protein exchange between bacterial species | Comparison with other ribosomal proteins |
| Dysbiosis investigation | L18 expression levels as biomarker | Response to dietary interventions | Correlation with other markers of microbial activity |
These applications exploit the specificity of B. thetaiotaomicron L18 to provide insights into the complex dynamics of the gut microbiome under different conditions, including health, disease, and therapeutic interventions .
What are the optimal conditions for studying B. thetaiotaomicron L18 interactions with other ribosomal components?
Investigating the interactions between B. thetaiotaomicron L18 and other ribosomal components requires carefully optimized experimental conditions that preserve native-like interaction properties. Based on research with ribosomal assembly systems, the following conditions have proven most effective:
| Parameter | Optimal Condition | Rationale | Monitoring Method |
|---|---|---|---|
| Buffer composition | 20 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgCl₂, 2 mM β-mercaptoethanol | Mimics intracellular environment, Mg²⁺ critical for RNA structure | Buffer optimization using thermal shift assays |
| Temperature | 30-37°C | Physiologically relevant, promotes dynamic interactions | Temperature-controlled fluorescence spectroscopy |
| RNA:protein ratio | 1:2 to 1:5 molar excess of protein | Ensures complete complex formation | Native gel electrophoresis |
| Incubation time | 30-60 minutes | Allows equilibrium to be reached | Time-course analysis by analytical ultracentrifugation |
| Avoiding degradation | Add RNase inhibitors, use DEPC-treated solutions | Prevents RNA degradation during experiments | RNA integrity analysis by capillary electrophoresis |
These conditions can be further adjusted for specific experimental techniques. For instance, structural studies using cryo-electron microscopy may require different buffer components to improve particle distribution and ice quality. The reconstitution of larger ribosomal subassemblies containing L18 typically benefits from a stepwise addition of components in the order that mimics the natural assembly pathway .
How can isotope labeling of recombinant B. thetaiotaomicron L18 be optimized for structural studies?
Isotope labeling of recombinant B. thetaiotaomicron L18 for structural studies by NMR spectroscopy or mass spectrometry requires specific adaptations to expression and purification protocols. The following approach has been optimized based on protocols for other ribosomal proteins:
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Expression medium selection:
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For ¹⁵N labeling: M9 minimal medium with ¹⁵NH₄Cl as sole nitrogen source
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For ¹³C labeling: M9 minimal medium with ¹³C-glucose as sole carbon source
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For deuteration: M9 prepared in D₂O with deuterated glucose
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Growth parameters:
| Labeling Type | Cell Density at Induction (OD₆₀₀) | IPTG Concentration | Temperature | Duration | Typical Yield Reduction |
|---|---|---|---|---|---|
| Single (¹⁵N) | 0.6-0.8 | 0.5 mM | 18°C | 16-20h | 20-30% |
| Double (¹⁵N/¹³C) | 0.8-1.0 | 0.5 mM | 18°C | 20-24h | 40-50% |
| Triple (¹⁵N/¹³C/²H) | 1.0-1.2 | 0.5 mM | 18°C | 24-30h | 60-70% |
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Adaptation strategies for improved yields:
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Stepwise adaptation to deuterated media for triple labeling
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Co-expression with chaperones for improved folding
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Supplementation with amino acid precursors in later growth phases
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Purification considerations:
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Maintain reducing conditions throughout purification
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Minimize exposure to proteases by using protease inhibitors
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Consider on-column refolding for proteins expressed in inclusion bodies
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The quality of isotope incorporation should be verified by mass spectrometry prior to structural studies, with expected incorporation rates of >95% for ¹⁵N, >90% for ¹³C, and >75% for deuterium under optimized conditions.
What methods are most effective for analyzing the impact of B. thetaiotaomicron L18 mutations on ribosome assembly?
Assessing the effects of B. thetaiotaomicron L18 mutations on ribosome assembly requires a multi-faceted approach that combines in vitro and in vivo methods. Based on studies of ribosomal assembly pathways, the following techniques provide complementary insights:
| Analytical Method | Information Provided | Advantages | Technical Considerations |
|---|---|---|---|
| Sucrose gradient ultracentrifugation | Distribution of ribosomal subunits and assembly intermediates | Quantitative analysis of multiple assembly states | Requires careful gradient preparation and fractionation |
| Quantitative mass spectrometry | Stoichiometry of ribosomal proteins in assembled particles | Precise protein composition data | Requires specialized equipment and software |
| Fluorescence-based ribosome assembly assays | Real-time monitoring of assembly kinetics | High sensitivity, less material required | Needs fluorescent labeling that doesn't interfere with assembly |
| Cryo-electron microscopy | Structural analysis of assembly intermediates | Direct visualization of structural defects | Resource-intensive, requires significant expertise |
| In vivo complementation assays | Functional impact of mutations | Physiologically relevant | Requires conditional expression systems |
For in vivo studies, complementation systems where wildtype L18 expression can be gradually reduced while mutant variants are expressed are particularly valuable. This approach enables assessment of both lethal and hypomorphic mutations. For the most comprehensive analysis, correlating structural defects observed in vitro with functional impacts measured in vivo provides the strongest evidence for the mechanistic role of specific L18 residues in ribosome assembly .
How can transcriptomics and proteomics be integrated to study the impact of L18 modification on B. thetaiotaomicron physiology?
Integrating transcriptomic and proteomic approaches provides a systems-level understanding of how L18 modifications affect B. thetaiotaomicron physiology. This multi-omics strategy reveals both direct translational effects and downstream adaptive responses:
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Experimental design considerations:
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Generate conditional L18 mutants or depletion strains
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Sample at multiple timepoints after L18 perturbation
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Include appropriate controls for general stress responses
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Data integration workflow:
| Stage | Transcriptomic Approach | Proteomic Approach | Integration Method | Expected Outcome |
|---|---|---|---|---|
| Primary data collection | RNA-seq | Label-free quantitative proteomics | Correlation analysis | Identification of concordant and discordant responses |
| Targeted validation | RT-qPCR for key genes | Targeted MS/MS for specific proteins | Direct comparison | Verification of key findings |
| Pathway analysis | Gene set enrichment analysis | Protein interaction networks | Pathway overlays | Identification of affected cellular processes |
| Translation efficiency | Ribosome profiling | Pulse-chase labeling | Translation efficiency calculation | Direct measurement of L18's impact on protein synthesis |
| Systems modeling | Network inference | Protein turnover analysis | Mathematical modeling | Predictive model of cellular response |
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Key cellular processes to monitor:
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Polysaccharide utilization capabilities
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Stress response pathways
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Cell envelope maintenance
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Central metabolism
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Virulence factor expression
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This integrated approach not only identifies differential gene and protein expression but also reveals post-transcriptional regulatory mechanisms affected by L18 modification. The correlation between transcriptome and proteome changes can highlight genes whose translation is particularly sensitive to alterations in ribosome composition, providing insights into specialized functions of L18 in B. thetaiotaomicron physiology .
How can aggregation issues during recombinant B. thetaiotaomicron L18 production be resolved?
Aggregation of recombinant B. thetaiotaomicron L18 during expression and purification is a common challenge that can significantly impact yield and functional studies. The following systematic troubleshooting approach addresses this issue:
| Problem Source | Solution Strategy | Implementation Details | Expected Outcome |
|---|---|---|---|
| Rapid expression rate | Reduce expression temperature | Lower to 16-18°C post-induction | Slower folding allowing proper conformation |
| Hydrophobic interactions | Modify buffer conditions | Add 0.1-0.5% non-ionic detergents (e.g., Triton X-100) | Disruption of non-specific hydrophobic interactions |
| Improper disulfide formation | Optimize redox environment | Include 1-5 mM DTT or BME in all buffers | Prevention of incorrect disulfide bonds |
| Co-aggregation with bacterial components | Increase purification stringency | Add intermediate ion exchange chromatography step | Removal of nucleic acids and other contaminants |
| Inherent structural properties | Co-express with chaperones | Use pGro7 (GroEL/ES) or pTf16 (trigger factor) | Assisted folding during expression |
| Concentration-dependent aggregation | Maintain dilute conditions | Keep below 1 mg/mL during purification steps | Prevention of concentration-dependent aggregation |
If aggregation persists despite these measures, alternative approaches including fusion partners (SUMO, MBP, or thioredoxin) that enhance solubility can be employed. The fusion partners can later be removed by proteolytic cleavage once the protein is properly folded. For long-term storage, adding 5-10% glycerol and flash-freezing in small aliquots can significantly reduce aggregation during freeze-thaw cycles .
What strategies help overcome challenges in obtaining enzymatically active recombinant B. thetaiotaomicron L18?
Ensuring enzymatic activity of recombinant B. thetaiotaomicron L18 for functional studies requires careful consideration of protein folding and maintenance of native structure. The following strategies have proven effective:
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Expression system optimization:
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Test multiple E. coli host strains (BL21, Rosetta, Origami)
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Consider low-temperature induction (16°C for 18-24 hours)
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Evaluate codon-optimized synthetic genes
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Buffer composition for maintaining activity:
| Buffer Component | Optimal Range | Functional Relevance | Monitoring Method |
|---|---|---|---|
| Magnesium ions | 5-10 mM MgCl₂ | Essential for RNA binding | Activity assays with varying [Mg²⁺] |
| Monovalent ions | 50-150 mM KCl or NaCl | Stabilizes electrostatic interactions | Thermal shift assays |
| pH | 7.0-7.5 | Maintains native charge distribution | pH-dependent activity profiling |
| Reducing agents | 1-2 mM DTT or TCEP | Prevents oxidation of critical cysteines | Comparison of reducing agent effectiveness |
| Stabilizing agents | 5% glycerol or 0.1M arginine | Prevents aggregation | Storage stability tests |
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Activity validation approaches:
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5S rRNA binding assays using filter binding or EMSA
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Integration into partial ribosomal reconstitution systems
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Structural integrity assessment by limited proteolysis
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Refolding strategies for inclusion body-derived protein:
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Gradient dialysis from denaturing conditions
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On-column refolding during affinity purification
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Pulsed dilution into refolding buffer
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By systematically addressing these factors, researchers can significantly improve the likelihood of obtaining enzymatically active recombinant L18 suitable for downstream functional and structural studies .
How should experimental design be adjusted to study B. thetaiotaomicron L18 under anaerobic conditions?
Studying B. thetaiotaomicron L18 under physiologically relevant anaerobic conditions presents unique challenges that require specific adaptations to standard experimental protocols. The following adjustments are recommended:
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Anaerobic experimental setup options:
| System Type | Components | Advantages | Limitations |
|---|---|---|---|
| Anaerobic chamber | Controlled atmosphere (N₂, CO₂, H₂), airlock, oxygen scrubber | Complete workflow in anaerobic environment | Expensive, limited equipment access |
| Hungate techniques | Specialized tubes, gas exchange system | Cost-effective, good for culture work | Limited manipulation options |
| Enzymatic oxygen scavenging | Glucose oxidase/catalase, pyranose oxidase systems | Compatible with microscopy, spectroscopy | Short-term anaerobicity only |
| Chemical reducing agents | Cysteine-HCl, sodium thioglycolate, titanium citrate | Simple implementation | May interfere with some assays |
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Critical adaptations for common techniques:
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Pre-reduce all media and buffers by degassing and adding reducing agents
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Use oxygen-impermeable materials (glass, specific plastics) and seal with butyl rubber stoppers
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Include resazurin (1-2 μg/mL) as an oxygen indicator in solutions
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Perform all manipulations in an anaerobic chamber or using strict anaerobic techniques
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Validation of anaerobic conditions:
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Monitor redox potential using redox electrodes
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Include oxygen-sensitive control proteins in activity assays
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Verify protein stability under reducing conditions using thermal shift assays
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Specific considerations for L18 studies:
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Assess RNA binding activity with and without oxygen exposure
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Compare protein-protein interactions under aerobic vs. anaerobic conditions
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Evaluate the impact of oxidative modifications on function
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These methodological adaptations ensure that the B. thetaiotaomicron L18 protein is studied under conditions that reflect its native environment in the gut, providing more physiologically relevant insights into its function and interactions .
How can CRISPR-Cas9 genome editing be applied to study L18 function in B. thetaiotaomicron?
CRISPR-Cas9 technology offers powerful approaches for investigating L18 function in B. thetaiotaomicron through precise genomic modifications. The following strategies have been developed:
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CRISPR-Cas9 modification systems for B. thetaiotaomicron:
| Approach | Delivery Method | Editing Efficiency | Application |
|---|---|---|---|
| pNBU2-based integration | Conjugation | Moderate (10-30%) | Site-specific mutations |
| λ-Red recombineering with CRISPR selection | Electroporation | High (40-60%) | Gene deletions, insertions |
| Plasmid-based expression | Conjugation | Variable (5-40%) | Conditional knockdowns |
| All-in-one CRISPR plasmid | Electroporation | Moderate (15-35%) | Multiplexed editing |
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Experimental designs for L18 functional studies:
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Creation of point mutations in conserved 5S rRNA binding residues
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Generation of conditional L18 depletion strains using inducible promoters
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Introduction of epitope tags for in vivo tracking and pull-down experiments
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Development of fluorescent protein fusions for localization studies
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Challenges specific to ribosomal protein gene editing:
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Essential nature of L18 requires conditional approaches
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Potential polar effects on adjacent genes in the operon
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Off-target effects requiring careful sgRNA design
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Maintaining physiological expression levels
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Validation strategies:
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RT-qPCR and Western blotting to confirm expression changes
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Growth curves to assess fitness impacts
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Ribosome profiling to evaluate translational effects
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Competition assays to measure relative fitness
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These CRISPR-based approaches enable unprecedented precision in manipulating L18 in its native context, allowing researchers to elucidate its role in B. thetaiotaomicron physiology, stress responses, and adaptation to the gut environment .
What are the prospects for using B. thetaiotaomicron L18 in synthetic biology applications?
The unique properties of B. thetaiotaomicron L18 present several opportunities for innovative synthetic biology applications, particularly in the context of engineered gut microbiome interventions:
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Potential synthetic biology applications:
| Application Area | Approach | Current Development Stage | Challenges |
|---|---|---|---|
| Engineered ribosomes | L18 modifications for altered translation | Proof-of-concept | Maintaining ribosome assembly with altered L18 |
| Biosensors | L18-based RNA binding domains | Early development | Specificity for target detection |
| Therapeutic delivery | L18 fusion proteins for gut targeting | Theoretical | Stability in GI tract |
| Metabolic engineering | Translation efficiency modulation | Preliminary studies | Balancing growth and production |
| Microbiome modulation | Engineered strains with modified L18 | Conceptual | In vivo competitive fitness |
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Key design principles for L18-based synthetic biology:
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Exploit the RNA-binding capacity of L18 for programmable interactions
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Utilize domains that maintain function outside the ribosomal context
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Leverage anaerobic adaptations for gut-specific applications
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Consider compatibility with the Bacteroides secretion systems
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Promising early applications:
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Development of tunable translation systems for controlled protein expression
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Creation of engineered B. thetaiotaomicron strains with modified stress responses
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Design of RNA-binding modules based on L18 structure for synthetic circuits
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The application of B. thetaiotaomicron L18 in synthetic biology represents an emerging frontier that combines fundamental ribosomal biology with practical applications in microbiome engineering, potentially leading to new therapeutic strategies for gut-associated disorders .
