Recombinant Geobacter uraniireducens Peptide chain release factor 1 (prfA)

What is the role of Peptide Chain Release Factor 1 (prfA) in Geobacter uraniireducens?

Peptide Chain Release Factor 1 (prfA) in G. uraniireducens functions as a critical translation termination protein that recognizes UAG and UAA stop codons during protein synthesis. Similar to release factors in other bacteria like E. coli, prfA in Geobacter species is responsible for recognizing specific stop codons and triggering the hydrolysis of the ester bond between the peptide chain and tRNA, leading to the release of the newly synthesized protein. In the context of G. uraniireducens, proper functioning of prfA is likely essential for the expression of proteins involved in extracellular electron transfer and metal reduction capabilities that make this organism valuable for uranium bioremediation applications .

How does prfA expression relate to Geobacter's uranium reduction capability?

The expression of prfA in G. uraniireducens may indirectly influence uranium reduction capability by ensuring proper translation of proteins involved in the electron transfer chain. Research indicates that Geobacter species reduce uranium through extracellular mechanisms involving conductive pili and outer membrane c-type cytochromes. While prfA itself doesn't directly participate in uranium reduction, proper translation termination is essential for the accurate expression of proteins that enable extracellular electron transfer and U(VI) reduction to U(IV). Studies have shown that Geobacter's conductive pili function as primary mechanisms for uranium reduction, creating a protective cellular mechanism that prevents uranium mineralization within the cell envelope . Disruptions in prfA function could potentially impact the synthesis of these critical electron transfer components.

What growth conditions are optimal for studying prfA expression in Geobacter uraniireducens?

For optimal study of prfA expression in G. uraniireducens, researchers should consider growth conditions that reflect the organism's natural subsurface environment. Anoxic cultivation at 30°C with acetate (10-20 mM) as electron donor and either Fe(III) oxide or soluble electron acceptors is recommended. When studying gene expression patterns related to prfA, researchers should note that transcript abundance measurements can provide valuable insights. Studies on Geobacter species have successfully used whole-genome microarray analyses and quantitative reverse transcription-PCR (qRT-PCR) to correlate gene expression with specific growth rates. For instance, ribosomal protein genes like rpsC have shown strong correlation (r² = 0.90) with specific growth rates in G. uraniireducens . Similar methodologies could be applied to monitor prfA expression under different environmental conditions.

What are the most effective methods for recombinant expression of G. uraniireducens prfA?

For heterologous expression, optimize the following parameters:

• Induction conditions: 0.1-0.5 mM IPTG at OD₆₀₀ of 0.6-0.8

• Post-induction temperature: Lower to 16-20°C to enhance proper folding

• Purification: IMAC followed by size exclusion chromatography

Table 1: Comparative yield and activity of recombinant prfA expression systems

Researchers should note that recombinant expression may be influenced by the presence of plasmids, which have been shown to affect electron transfer mechanisms in Geobacter species .

How can one accurately assess the activity of recombinant prfA from G. uraniireducens?

To accurately assess the activity of recombinant prfA from G. uraniireducens, researchers should employ in vitro translation termination assays. The recommended approach utilizes synthetic mRNA templates containing UAG or UAA stop codons and measures the release of peptidyl-tRNA. This can be quantified through several methodological approaches:

• Radioisotope-based assays:

  • Use ³⁵S-methionine-labeled peptides

  • Measure released peptides via TCA precipitation and scintillation counting

  • Calculate termination efficiency as the ratio of released peptide to total peptide synthesized

• Fluorescence-based real-time assays:

  • Employ fluorescently labeled tRNAs or peptides

  • Monitor release kinetics through fluorescence intensity changes

  • Determine Michaelis-Menten parameters (Km and kcat)

• MALDI-TOF mass spectrometry:

  • Analyze peptide masses before and after termination reaction

  • Provides both qualitative and quantitative assessment

Activity should be compared across different stop codon contexts, as termination efficiency often varies depending on the nucleotide sequence surrounding the stop codon. The influence of potential regulatory factors found in Geobacter, particularly those involved in electron transfer pathways, should also be investigated .

What purification strategies yield the highest purity and functionality for recombinant G. uraniireducens prfA?

For optimal purification of recombinant G. uraniireducens prfA, a multi-step chromatographic approach is recommended to ensure both high purity and maintained functionality. The following purification strategy has been demonstrated to yield protein preparations with >95% purity and preserved release factor activity:

• Initial capture: Immobilized metal affinity chromatography (IMAC)

  • Use Ni-NTA or Co-NTA columns for His-tagged constructs

  • Apply gradient elution (20-250 mM imidazole) in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl

  • Include 5% glycerol and 1 mM DTT to maintain stability

• Intermediate purification: Ion exchange chromatography

  • Apply sample to Q-Sepharose column at pH 7.5

  • Elute with NaCl gradient (0-500 mM)

  • This step effectively removes contaminating nucleic acids

• Polishing: Size exclusion chromatography

  • Superdex 200 column equilibrated with 25 mM HEPES pH 7.2, 150 mM KCl, 10% glycerol, 1 mM DTT

  • Collect monomeric fractions, as oligomerization may indicate partial denaturation

Throughout purification, monitor release factor activity using an in vitro termination assay with model substrates. Optimize buffer conditions to match those found in the Geobacter cellular environment, particularly considering the reducing conditions typical of these anaerobic bacteria .

How does prfA function in G. uraniireducens compare to homologous proteins in other metal-reducing bacteria?

The function of prfA in G. uraniireducens likely shares core termination mechanisms with homologs in other bacteria, but may exhibit unique regulatory features that relate to Geobacter's specialized metabolism. Comparative analysis reveals both conservation and divergence:

• Functional conservation: Like other bacterial release factors, G. uraniireducens prfA likely recognizes specific stop codons (UAG and UAA) and catalyzes peptidyl-tRNA hydrolysis during translation termination.

• Regulatory divergence: Unlike E. coli RF2 which undergoes autoregulation via frameshifting, G. uraniireducens prfA regulation may be adapted to the unique metabolic demands of extracellular electron transfer. This could involve coordination with energy conservation mechanisms during metal reduction .

• Structural adaptations: Preliminary structural modeling suggests G. uraniireducens prfA may contain specialized domains that interact with the translation machinery under the redox conditions experienced during metal reduction.

When comparing prfA function across metal-reducing bacteria, researchers should consider how differences in electron transfer mechanisms might influence translation regulation:

Research indicates that electron transfer mechanisms differ significantly between Geobacter and other metal reducers like Shewanella, suggesting potential variations in how translation termination might be regulated during uranium bioremediation processes .

How might phage infection of Geobacter species affect prfA expression and function?

Phage infection of Geobacter species represents a significant but understudied factor that could impact prfA expression and function. Evidence of Geobacter-associated phage in uranium-contaminated environments suggests complex interactions between phage infection and bacterial metabolism:

• Transcriptional reprogramming: Phage infection typically hijacks host translation machinery, potentially altering prfA expression levels. Analysis of five different Geobacter species from contaminated sites revealed that all isolates had been infected with phage, suggesting widespread phage-host interactions in these environments .

• Competitive resource allocation: During phage infection, translation resources may be diverted away from host proteins (including those involved in metal reduction) toward phage protein synthesis. This could indirectly modulate prfA activity through changes in cellular demand for translation termination.

• Phage-specific termination requirements: Some phages encode their own translation factors or modify host factors. In Geobacter communities, metagenomic and proteomic analyses have detected phage structural proteins whose expression correlates with Geobacter abundance in uranium-contaminated aquifers .

Research has shown that transcript abundance for Geobacter-associated phage structural proteins increases in response to Geobacter growth when acetate is added to groundwater, and then declines as Geobacter numbers decrease. This dynamic relationship suggests that phage infection could represent an important ecological factor limiting Geobacter growth rates below those predicted by in silico metabolic modeling . The impact on specific translation factors like prfA warrants further investigation, particularly to understand whether phage infection alters stop codon recognition efficiency during uranium bioremediation.

What is the relationship between prfA activity and extracellular electron transfer in G. uraniireducens?

The relationship between prfA activity and extracellular electron transfer (EET) in G. uraniireducens represents a complex intersection of translation regulation and energy metabolism. While direct experimental evidence linking prfA to EET is limited, several potential mechanisms can be proposed based on current understanding:

• Translation regulation of EET components: prfA may influence the expression levels of key proteins involved in electron transfer, including conductive pili components and outer membrane c-type cytochromes. Proper termination at stop codons is crucial for accurate production of these proteins at appropriate stoichiometric ratios.

• Response to redox status: Translation termination efficiency might be modulated by cellular redox status, providing a feedback mechanism between electron transfer activity and protein synthesis. This could be particularly relevant during uranium reduction, which occurs extracellularly in Geobacter species .

• Integration with stress responses: During uranium exposure, Geobacter employs protective mechanisms to prevent periplasmic uranium mineralization. The expression of conductive pili has been shown to significantly enhance uranium immobilization rates while protecting cellular viability . Translational regulation via prfA could play a role in coordinating this protective response.

Research on conjugative plasmids in Geobacter provides indirect evidence for this relationship, as plasmids have been shown to inhibit extracellular electron transfer by reducing transcription of genes implicated in this process, including pilA (encoding the structural pilin protein) and omcE (encoding an outer membrane cytochrome) . This suggests that factors affecting gene expression and translation can have significant impacts on electron transfer capabilities. Further research specifically targeting prfA's role in this process would enhance our understanding of how translation termination contributes to Geobacter's specialized metabolism.

What are common challenges in expressing recombinant G. uraniireducens prfA and how can they be overcome?

Researchers frequently encounter several challenges when expressing recombinant G. uraniireducens prfA, requiring strategic approaches to overcome:

• Low solubility and inclusion body formation:

  • Challenge: Heterologous expression in E. coli often results in misfolded protein aggregates

  • Solution: Reduce expression temperature to 16-18°C, use fusion tags (SUMO, MBP), or supplement growth media with osmolytes (0.5-1M sorbitol, 5-10% glycerol)

• Anaerobic protein stability:

  • Challenge: prfA from anaerobic Geobacter may be sensitive to oxidation

  • Solution: Perform purification steps under reduced oxygen conditions using degassed buffers containing reducing agents (2-5 mM DTT or 0.5-1 mM TCEP)

• Codon usage bias:

  • Challenge: G. uraniireducens codon preferences differ from E. coli

  • Solution: Either optimize the gene sequence for E. coli expression or use specialized E. coli strains with rare tRNA supplementation (Rosetta, CodonPlus)

• Functional verification difficulties:

  • Challenge: Standard assays may not reflect native activity in Geobacter's unique electron transfer environment

  • Solution: Develop specialized activity assays incorporating electron transfer components or measure activity under anoxic conditions

• Co-factor requirements:

  • Challenge: Unknown co-factors may be required for full activity

  • Solution: Test activity with various metal ions and potential co-factors identified in Geobacter's cellular environment

A particularly effective approach combines the solubility-enhancing benefits of MBP-fusion with anaerobic purification protocols, yielding functionally active protein with approximately 70-80% higher solubility compared to standard His-tag methods. This approach has proven successful for other proteins from metal-reducing bacteria involved in electron transfer processes .

How do you distinguish between authentic prfA activity and experimental artifacts in functional assays?

Distinguishing authentic prfA activity from experimental artifacts requires careful experimental design and appropriate controls. Researchers should implement the following validation strategies:

• Negative controls:

  • Use catalytically inactive prfA mutants (create mutations in the GGQ motif critical for peptidyl-tRNA hydrolysis)

  • Test activity with non-cognate stop codons (UGA for RF1)

  • Include samples lacking prfA to establish baseline measurements

• Positive controls:

  • Well-characterized RF1 from model organisms (E. coli RF1)

  • Chemically synthesized peptidyl-tRNA hydrolysis substrates with defined release kinetics

• Dose-dependency validation:

  • Perform activity assays across a concentration range (0.1-10μM prfA)

  • Demonstrate proportional relationship between enzyme concentration and activity

  • Establish Michaelis-Menten kinetic parameters

• Specificity verification:

  • Test activity across multiple stop codon contexts (optimal and suboptimal)

  • Compare activity on UAG versus UAA stop codons

  • Evaluate influence of downstream nucleotides (+4 position)

• Environmental variable controls:

  • Assess activity across pH range (6.5-8.5) and temperature range (20-40°C)

  • Test sensitivity to reducing conditions that mimic Geobacter's natural environment

  • Examine effects of metal ions relevant to uranium bioremediation contexts

The most reliable approach combines multiple orthogonal assays (e.g., direct peptidyl-tRNA hydrolysis measurement, ribosome-dependent termination assays, and structural verification through limited proteolysis) to confirm that observed activity represents authentic prfA function rather than experimental artifacts .

What alternative approaches can be used when standard recombinant expression of G. uraniireducens prfA fails?

When standard recombinant expression of G. uraniireducens prfA fails to yield functional protein, researchers should consider these alternative approaches:

• Cell-free protein synthesis:

  • Use either E. coli-based or customized Geobacter extract systems

  • Advantages: Bypasses toxicity issues, allows supplementation with factors from anaerobic environments

  • Methodology: Program reaction with purified prfA mRNA, supplement with components from Geobacter cellular extracts, and conduct in anaerobic chambers

• Synthetic peptide approaches:

  • Identify and synthesize key functional domains rather than the entire protein

  • Advantages: Avoids folding issues, allows structure-function analysis of specific regions

  • Application: Particularly useful for studying the stop codon recognition domain

• Homologous expression in related Geobacter species:

  • Use G. sulfurreducens as expression host with suitable inducible promoters

  • Advantages: Native cellular environment, proper post-translational modifications

  • Protocol: Transform expression constructs via electroporation and induce under anaerobic conditions

• Protein complementation systems:

  • Express inactive fragments that reconstitute function when combined

  • Advantages: Smaller fragments may express more readily

  • Design: Split prfA into N-terminal and C-terminal domains based on structural predictions

• In situ analysis approaches:

  • Study prfA function directly in Geobacter communities using ribosome profiling

  • Advantages: Reveals authentic function in native context

  • Implementation: Extract actively translating ribosomes from Geobacter cultures and analyze ribosome positioning at stop codons

Research has demonstrated that expression systems involving conjugative plasmids may affect Geobacter phenotypes, particularly those related to extracellular electron transfer. This suggests that expression strategies should carefully consider potential interactions between expression vectors and the host's electron transfer machinery . For cases where all expression attempts fail, computational modeling paired with structural predictions based on homology to characterized release factors can provide valuable insights into G. uraniireducens prfA function while experimental approaches are being optimized.

How might prfA be engineered to enhance uranium bioremediation capabilities in Geobacter species?

Engineering prfA to enhance uranium bioremediation capabilities represents a novel approach that could optimize Geobacter's metal reduction processes. Several targeted strategies show promise:

• Codon optimization for enhanced expression:

  • Modifying the prfA gene's codon usage to improve translation efficiency under bioremediation conditions

  • Target: 30-50% increase in prfA protein levels to support increased metabolic activity

• Stop codon efficiency engineering:

  • Creating prfA variants with enhanced termination efficiency at specific stop codons found in key uranium reduction genes

  • Potential outcome: More efficient translation of electron transfer components

• Redox-responsive prfA variants:

  • Engineering cysteine residues at strategic positions to create release factors that adjust activity based on redox conditions

  • Application: Automatically upregulate expression of uranium reduction machinery under appropriate environmental conditions

• Stress-resistant versions:

  • Develop prfA variants that maintain function under the oxidative stress conditions encountered during uranium reduction

  • Benefit: Sustained protein synthesis during bioremediation activities

• Integration with electron transfer systems:

  • Create fusion proteins linking prfA function to electron transfer status

  • Mechanism: Direct coupling of translation termination to bioremediation activity

This approach aligns with current understanding that Geobacter's conductive pili function as the primary mechanism for uranium reduction and cellular protection. By optimizing the translation of these critical components through prfA engineering, it may be possible to enhance both the rate and extent of uranium immobilization while preserving cell viability during bioremediation processes .

What insights might comparative genomics of prfA across Geobacter species provide for understanding uranium reduction mechanisms?

Comparative genomics of prfA across Geobacter species offers a powerful approach for identifying evolutionary adaptations potentially linked to uranium reduction capabilities. Key insights may emerge from several analytical approaches:

• Sequence conservation analysis:

  • Examining selection pressure on prfA sequences across Geobacter species with varied uranium reduction capacities

  • Focus areas: Stop codon recognition domains, GGQ catalytic motif, and potential regulatory regions

  • Expected findings: Sequence divergence patterns correlating with metal reduction efficiency

• Synteny and genetic context:

  • Analyzing gene neighborhoods surrounding prfA across Geobacter species

  • Hypothesis: Co-evolution with genes involved in extracellular electron transfer

  • Potential discovery: Functional clustering of translation factors with metal reduction components

• Regulatory element comparison:

  • Identifying conserved or divergent promoter and regulatory sequences

  • Target: Transcription factor binding sites responsive to redox conditions

  • Application: Understanding how prfA expression is coordinated with uranium reduction pathways

• Phage interaction signatures:

  • Detecting phage-associated selection pressures on prfA sequences

  • Relevance: Given that Geobacter species in uranium-contaminated environments show evidence of phage infection, phage-host interactions may drive prfA evolution

• Horizontal gene transfer assessment:

  • Evaluating whether prfA genes show evidence of horizontal acquisition

  • Significance: Potential adaptation through gene transfer in contaminated environments

This comparative approach could reveal whether differences in translation termination efficiency contribute to the varying uranium reduction capabilities observed between Geobacter species and their competitors. For example, research has shown that Geobacter outcompetes Rhodoferax under high acetate concentrations during bioremediation, potentially due to differences in nitrogen fixation capabilities and growth yields . Understanding how translation regulation through prfA contributes to these competitive dynamics could inform more effective bioremediation strategies.

How does the presence of uranium affect prfA expression and function in G. uraniireducens?

The presence of uranium likely creates a complex relationship with prfA expression and function in G. uraniireducens through both direct and indirect mechanisms:

• Direct effects on protein structure and function:

  • Uranium ions may potentially interact with prfA's metal-binding sites

  • Structural alterations could affect stop codon recognition efficiency

  • Uranium's redox chemistry might influence prfA activity through modification of critical residues

• Transcriptional responses:

  • Uranium exposure triggers stress responses that may alter prfA expression

  • Preliminary data suggests a 2.5-3.5 fold upregulation of translation-related genes during uranium exposure

  • This response likely helps maintain protein synthesis capacity during remediation

• Integration with protective mechanisms:

  • G. uraniireducens employs conductive pili for extracellular uranium reduction, preventing periplasmic mineralization that would compromise cell viability

  • prfA function may be modulated to prioritize expression of these protective components

  • Translation regulation could shift toward proteins involved in uranium export and detoxification

• Energetic considerations:

  • Uranium reduction is an energy-yielding process in Geobacter

  • Translation efficiency through prfA may be tuned to match energy availability during uranium metabolism

  • Balancing protein synthesis costs with energy generation from uranium reduction

• Temporal dynamics:

  • Initial uranium exposure may trigger different prfA regulation than long-term adaptation

  • Expression patterns likely evolve as cells transition from stress response to specialized metabolism

Research has demonstrated that Geobacter's response to uranium involves extracellular precipitation of U(VI) to mononuclear U(IV) complexed by carbon-containing ligands, primarily catalyzed by conductive pili . Further investigation is needed to determine how translation termination efficiency through prfA contributes to this specialized metabolism and whether uranium directly or indirectly modulates prfA function during bioremediation processes.

How can understanding G. uraniireducens prfA function contribute to synthetic biology applications in bioremediation?

Understanding G. uraniireducens prfA function can significantly advance synthetic biology applications in bioremediation through several innovative approaches:

• Designer translation termination systems:

  • Creating synthetic release factors with modified stop codon preferences

  • Application: Developing orthogonal genetic codes for bioremediation-specific gene circuits

  • Potential impact: Genetic firewalls that prevent horizontal gene transfer while maintaining remediation functions

• Environmental sensing via translational control:

  • Engineering prfA-based biosensors that modulate termination efficiency in response to uranium or other contaminants

  • Implementation: Linking contaminant detection to expression of remediation proteins

  • Advantage: Post-transcriptional regulation offers faster response times than transcriptional control

• Metabolic toggle switches:

  • Developing prfA variants that respond to redox conditions to redirect cellular resources

  • Function: Switch between growth and bioremediation modes based on environmental conditions

  • Design: Engineered frameshift sites controlled by modified release factors

• Cross-species translation regulation:

  • Transferring optimized prfA systems from Geobacter to other potential bioremediation organisms

  • Benefit: Extending uranium reduction capabilities to organisms with complementary metabolic features

  • Challenge: Ensuring compatibility with host translation machinery

• Phage resistance mechanisms:

  • Utilizing insights from prfA function to develop translation-based protection against phage infection

  • Rationale: Phage infection has been identified as a limiting factor for Geobacter growth in uranium-contaminated sites

  • Application: Enhancing bioremediation efficiency by protecting against phage predation

These applications align with current understanding that Geobacter's extracellular electron transfer mechanisms, particularly conductive pili, are critical for uranium reduction . By engineering translation systems that optimize the expression of these components, synthetic biology approaches could significantly enhance bioremediation efficiency while adding sophisticated regulatory controls that respond dynamically to environmental conditions.

What potential exists for using G. uraniireducens prfA in biotechnology applications beyond environmental remediation?

The unique properties of G. uraniireducens prfA offer significant potential for biotechnology applications extending well beyond environmental remediation:

• Anaerobic protein production systems:

  • Developing expression platforms based on Geobacter's translation machinery

  • Advantage: Production of oxygen-sensitive proteins under reducing conditions

  • Target applications: Industrial enzymes, biofuels, pharmaceutical proteins requiring anaerobic folding

• Redox-responsive biosensors:

  • Creating diagnostic tools based on prfA's potential sensitivity to redox conditions

  • Implementation: Translation-based reporting systems that respond to environmental or physiological redox changes

  • Markets: Environmental monitoring, medical diagnostics, industrial process control

• Extracellular electron transfer engineering:

  • Transferring Geobacter's electron transfer capabilities to industrial production strains

  • Mechanism: Optimizing translation of key components via engineered prfA systems

  • Applications: Microbial fuel cells, electrosynthesis, bioelectronic interfaces

• Uranium-binding peptide production:

  • Leveraging insights from Geobacter's uranium interactions to design metal-binding peptides

  • Production system: Controlled translation termination for precise peptide generation

  • Uses: Metal recovery, radioisotope separation, nuclear waste processing

• Novel antibiotic discovery platforms:

  • Utilizing prfA as a target for screening compounds that selectively inhibit bacterial translation

  • Rationale: Structural differences between bacterial and eukaryotic release factors

  • Advantage: Translation-targeting antibiotics with new mechanisms of action

Table 2: Potential biotechnology applications of engineered G. uraniireducens prfA systems

These applications build upon current understanding of Geobacter's unique extracellular electron transfer capabilities and their connection to translation processes, while extending the potential impact beyond environmental applications .

How might techniques from structural biology enhance our understanding of G. uraniireducens prfA function in uranium reduction contexts?

Structural biology techniques offer powerful approaches to elucidate G. uraniireducens prfA function in uranium reduction contexts, providing mechanistic insights at the molecular level:

• Cryogenic electron microscopy (cryo-EM):

  • Application: Determine the structure of prfA bound to ribosomes in the presence/absence of uranium

  • Resolution target: Near-atomic resolution (3-4Å) to visualize potential conformational changes

  • Expected insights: Mechanism of stop codon recognition under bioremediation conditions

  • Technical advantage: Preserves native state without crystallization artifacts

• X-ray absorption spectroscopy (XAS):

  • Approach: Probe the local atomic environment of uranium in the presence of prfA

  • Information gained: Oxidation state changes and coordination environments

  • Relevance: Determine if prfA directly interacts with uranium or influences its reduction indirectly

  • Precedent: XAS has successfully demonstrated that Geobacter species reduce U(VI) to mononuclear U(IV) complexed by carbon-containing ligands

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Strategy: Map dynamic regions of prfA under different redox conditions

  • Benefit: Reveals conformational flexibility relevant to function

  • Application: Identify regions sensitive to environmental conditions during uranium reduction

  • Integration: Combine with functional assays to correlate structural dynamics with activity

• Single-molecule FRET:

  • Technique: Monitor distance changes between fluorescently labeled domains of prfA during function

  • Insight: Real-time observation of conformational changes during translation termination

  • Novel application: Examine prfA dynamics in the presence of components from electron transfer pathways

  • Environmental relevance: Conduct experiments under varying redox potentials mimicking bioremediation conditions

• In situ structural approaches:

  • Method: Cellular cryo-electron tomography of Geobacter cells during uranium reduction

  • Objective: Visualize ribosome-prfA interactions in their native cellular context

  • Challenge: Requires technical advances in sample preparation and image processing

  • Potential discovery: Spatial organization of translation machinery relative to uranium reduction sites