Recombinant Staphylococcus aureus Uncharacterized N-acetyltransferase SAS1110 (SAS1110)

Abstract

This comprehensive FAQ collection addresses fundamental and advanced research questions regarding the uncharacterized N-acetyltransferase SAS1110 in Staphylococcus aureus. While SAS1110 remains largely uncharacterized, this resource synthesizes existing knowledge and provides methodological guidance for researchers investigating this protein. The collection distinguishes between established information and research hypotheses, offering experimental strategies for functional characterization and potential biological roles in S. aureus metabolism and pathogenesis.

Basic Characterization of SAS1110

What is currently known about the uncharacterized N-acetyltransferase SAS1110 in Staphylococcus aureus?

SAS1110 is annotated as an uncharacterized N-acetyltransferase in Staphylococcus aureus genomes. It belongs to the N-acetyltransferase family of enzymes (EC 2.3.1.-) that catalyze the transfer of acetyl groups from acetyl-CoA to various substrates . The protein is referenced in multiple S. aureus genome databases including comparative genomic analyses of different S. aureus strains . Genomic data indicates SAS1110 is conserved across multiple S. aureus strains with homologous genes identified as SAR1152 in strain MRSA252, NWMN_1086 in strain Newman, and SAV1176 in strain Mu50 .

Despite being cataloged in genomic databases, SAS1110 remains functionally uncharacterized, with limited experimental validation of its enzymatic activity, substrate specificity, or biological role in S. aureus. Current annotation is primarily based on sequence homology to other acetyltransferases, with GO molecular function annotation suggesting transferase activity, specifically transferring acyl groups (GO:0016746) .

How should researchers approach the functional characterization of an uncharacterized protein like SAS1110?

Characterizing uncharacterized proteins requires a systematic experimental approach:

• Recombinant protein expression and purification

  • Express SAS1110 in multiple heterologous systems (E. coli, yeast, baculovirus, mammalian cells) to determine optimal expression conditions

  • Purify to >85% homogeneity via affinity chromatography followed by size exclusion or ion exchange chromatography

• Bioinformatic analysis

  • Conduct thorough phylogenetic analysis to identify close homologs with known functions

  • Predict structural features and potential substrate binding sites through homology modeling

• Enzymatic activity screening

  • Develop in vitro assays to test N-acetyltransferase activity against candidate substrates

  • Screen compound libraries focused on:

    • Sulfur-containing metabolites (cysteine, N-acetylcysteine, homocysteine)

    • Antibiotics known to undergo acetylation

    • Metabolic intermediates in relevant S. aureus pathways

• Gene knockout/knockdown studies

  • Generate SAS1110 deletion mutants in S. aureus to observe phenotypic changes

  • Employ experimental designs similar to those used for studying S. aureus cystine transporter systems

• Transcriptomic and proteomic analyses

  • Compare gene expression profiles between wild-type and mutant strains

  • Identify conditions that upregulate SAS1110 expression

This multi-faceted approach allows researchers to systematically characterize the function of uncharacterized proteins like SAS1110, moving beyond genomic annotation to functional understanding.

What sequence-based predictions can be made about SAS1110's potential function?

Based on sequence analysis and homology to characterized N-acetyltransferases, several predictions can be made about SAS1110:

• Catalytic mechanism: SAS1110 likely catalyzes the transfer of acetyl groups from acetyl-CoA to specific substrates, functioning similarly to other bacterial N-acetyltransferases.

• Domain architecture: Sequence analysis suggests the presence of a conserved N-acetyltransferase domain with the GNAT (GCN5-related N-acetyltransferase) fold, containing conserved motifs for acetyl-CoA binding and substrate recognition.

• Potential substrate classes:

  • Given S. aureus biology, potential substrates may include:

    • Sulfur-containing amino acids or metabolites (cysteine, homocysteine)

    • N-acetylcysteine (NAC), which is utilized as a sulfur source in S. aureus

    • Antibiotics that can be inactivated by acetylation

• Functional context:

  • May participate in sulfur metabolism pathways, similar to the role of TcyABC and TcyP systems in S. aureus

  • Could potentially contribute to stress responses or adaptation to nutrient limitation

These predictions provide a starting point for experimental investigation but require validation through the methodological approaches described above.

Experimental Approaches for SAS1110 Research

What expression systems are most suitable for recombinant production of SAS1110?

Multiple expression systems have been used successfully for recombinant S. aureus proteins, each with specific advantages for different research applications:

For SAS1110 specifically, commercial sources have successfully produced the recombinant protein in all four systems with >85% purity (SDS-PAGE). When selecting an expression system, researchers should consider:

• Research objectives: Structural studies may prioritize yield (E. coli), while functional studies may require proper folding (yeast/baculovirus).

• Protein characteristics: The presence of disulfide bonds or required PTMs should influence system choice.

• Downstream applications: Crystallography requires high purity and homogeneity, while enzymatic assays require properly folded, active protein.

• Optimization strategies: For each system, optimize:

  • Codon usage for the expression host

  • Induction conditions (temperature, inducer concentration)

  • Fusion tags to enhance solubility (His, GST, MBP)

The most effective approach often involves testing multiple expression systems in parallel to identify optimal conditions for specific research objectives.

What methodological approaches can determine substrate specificity of SAS1110?

Determining substrate specificity for an uncharacterized N-acetyltransferase requires a multi-faceted experimental approach:

• Candidate substrate screening:

  • Test activity against a panel of potential substrates based on:

    • Known substrates of characterized N-acetyltransferases

    • Metabolites in S. aureus-relevant pathways

    • Sulfur-containing compounds like cysteine and N-acetylcysteine

  • Monitor acetylation using:

    • HPLC-based detection of acetylated products

    • Colorimetric assays tracking CoA-SH release

    • Mass spectrometry to identify modified substrates

• Kinetic characterization:

  • For identified substrates, determine:

    • Km and Vmax values

    • Substrate concentration ranges

    • Optimal reaction conditions (pH, temperature, cofactors)

• Structure-based approaches:

  • Crystallize SAS1110 with potential substrates/substrate analogs

  • Perform molecular docking studies

  • Use site-directed mutagenesis to validate predicted binding residues

• Activity-based protein profiling:

  • Develop chemical probes to identify enzyme-substrate interactions

  • Use clickable acetyl-CoA analogs to track acetylation events

• Metabolomic approaches:

  • Compare metabolite profiles between wild-type and SAS1110 knockout strains

  • Identify accumulated precursors or depleted products

This systematic approach allows researchers to move from initial substrate identification to detailed characterization of enzyme specificity and catalytic properties.

How can researchers design experimental controls when studying an uncharacterized enzyme like SAS1110?

• Negative controls:

  • Heat-inactivated enzyme preparations

  • Catalytically inactive mutants (generated by site-directed mutagenesis of predicted active site residues)

  • Reaction mixtures lacking essential components (substrate, cofactor, enzyme)

• Positive controls:

  • Well-characterized N-acetyltransferases with known activity

  • For S. aureus studies, include characterized acetyltransferases when available

  • Chemical acetylation reactions to generate reference standards

• Substrate specificity controls:

  • Structurally similar non-substrate compounds

  • Substrate analogs with modified reactive groups

  • Competitive inhibitors when identified

• Genetic complementation controls:

  • When studying SAS1110 knockout phenotypes, include:

    • Wild-type strain

    • Knockout strain

    • Complemented strain (knockout with plasmid-encoded SAS1110)

    • Strain expressing catalytically inactive SAS1110

• Experimental design considerations:

  • Randomization of sample processing

  • Blinding of sample identity during analysis

  • Technical and biological replicates

  • Statistical power calculations for determining appropriate sample sizes

These control strategies help distinguish true enzymatic activity from artifacts and establish the specificity of observed functions, critical for characterizing previously unstudied proteins like SAS1110.

SAS1110 in S. aureus Biology and Metabolism

How might SAS1110 integrate with known S. aureus metabolic pathways?

Based on its classification as an N-acetyltransferase, SAS1110 could potentially integrate with several key metabolic pathways in S. aureus:

• Sulfur metabolism:

  • S. aureus has specific transporters (TcyABC and TcyP) for acquiring cystine and cysteine as sulfur sources

  • SAS1110 could potentially acetylate cysteine-derived metabolites, affecting:

    • Utilization of N-acetylcysteine (NAC) as a sulfur source

    • Detoxification of reactive sulfur species

    • Biosynthesis of sulfur-containing cofactors

• Amino acid metabolism:

  • N-acetyltransferases often modify amino acids or amino acid derivatives

  • Could regulate amino acid pools through acetylation/deacetylation cycles

  • May participate in specialized amino acid modifications specific to S. aureus

• Antibiotic resistance mechanisms:

  • Some N-acetyltransferases modify antibiotics, reducing their efficacy

  • Could potentially contribute to resistance against certain classes of antibiotics

  • May acetylate xenobiotics for detoxification

• Cell wall metabolism:

  • S. aureus cell wall components include modified amino acids

  • SAS1110 might participate in acetylation steps during peptidoglycan synthesis or modification

To experimentally investigate these possibilities, researchers could:

• Compare metabolite profiles between wild-type and SAS1110 mutant strains grown with different sulfur sources

• Test growth phenotypes in media with various sole sulfur sources, similar to experimental approaches used for TcyABC and TcyP characterization

• Perform transcriptomic analysis to identify co-regulated genes that might indicate metabolic pathway associations

What role might SAS1110 play in S. aureus virulence or pathogenesis?

While the specific role of SAS1110 in virulence remains uncharacterized, several hypotheses can be proposed based on the functions of N-acetyltransferases in bacterial pathogenesis:

To investigate these possibilities, researchers should consider:

• Infection models comparing wild-type and SAS1110 knockout strains

• Biofilm formation assays under various conditions

• Survival assays under different stress conditions

• Transcriptomic analysis during infection or stress conditions to determine if SAS1110 is upregulated

These approaches would help elucidate whether SAS1110 contributes to S. aureus virulence and through what specific mechanisms.

Could SAS1110 be involved in cysteine metabolism or utilization in S. aureus?

The involvement of SAS1110 in cysteine metabolism is a compelling research hypothesis based on several lines of evidence:

• S. aureus sulfur acquisition systems:

  • S. aureus possesses specific transporters (TcyABC and TcyP) for acquiring cystine and cysteine

  • These transporters are required for growth in media with cysteine or N-acetylcysteine (NAC) as the sole sulfur source

  • N-acetyltransferases could logically function downstream of these transport systems

• Potential metabolic roles:

  • Acetylation of cysteine to form N-acetylcysteine (NAC)

  • Deacetylation of NAC to release free cysteine for metabolism

  • Modification of cysteine-containing peptides or proteins

• Experimental evidence from related systems:

  • S. aureus can utilize N-acetylcysteine as a sulfur source

  • N-acetyltransferases in other bacteria modify sulfur-containing amino acids

  • SAS1110 could potentially form part of the metabolic pathway for NAC utilization

To investigate this hypothesis, researchers could:

• Compare growth phenotypes:

  • Culture wild-type and SAS1110 knockout strains in defined media with different sulfur sources:

    • Cysteine

    • Cystine

    • N-acetylcysteine

    • Homocysteine/homocystine

• Metabolite profiling:

  • Track the fate of isotope-labeled cysteine or NAC in wild-type vs. knockout strains

  • Identify accumulation or depletion of specific metabolites

• Enzymatic assays:

  • Test SAS1110's ability to acetylate cysteine in vitro

  • Assess activity with related sulfur compounds

• Gene expression analysis:

  • Determine if SAS1110 is co-regulated with known sulfur metabolism genes

  • Examine expression patterns under sulfur limitation, similar to studies showing upregulation of tcyP and tcyA during sulfur starvation

This systematic approach would help determine whether SAS1110 functions in cysteine metabolism and define its specific role in this pathway.

Advanced Research Directions and Experimental Design

What transcriptomic and proteomic approaches would be most informative for studying SAS1110 function?

Integrating multiple omics approaches can provide comprehensive insights into SAS1110 function:

• Transcriptomic approaches:

  • RNA-seq comparison of wild-type and ΔsasA1110 strains under:

    • Standard growth conditions

    • Sulfur limitation (to identify condition-specific effects)

    • Various stress conditions (oxidative, antibiotic, etc.)

  • Ribosome profiling to examine translational impacts, similar to the Ribo-seq approach used to study Nat10 regulation

  • Time-course expression analysis during growth phases or infection models

• Proteomic approaches:

  • Global acetylome analysis comparing wild-type and knockout strains to identify potential substrates

  • Protein-protein interaction studies using:

    • Co-immunoprecipitation with tagged SAS1110

    • Proximity labeling methods (BioID, APEX)

    • Yeast two-hybrid screening

  • Quantitative proteomics to identify proteins with altered abundance in knockout strains

• Integrative approaches:

  • Multi-omics integration to correlate transcriptional, translational, and protein-level changes

  • Network analysis to position SAS1110 within S. aureus metabolic or regulatory networks

  • Comparative analysis across multiple S. aureus strains to identify strain-specific functions

• Experimental design considerations:

  • Include appropriate time points capturing both immediate and adaptive responses

  • Use targeted approaches to validate high-throughput findings

  • Consider both laboratory and infection-relevant conditions

These approaches would generate testable hypotheses about SAS1110 function and identify potential substrates or interacting partners for further investigation.

How can researchers design definitive experiments to determine if SAS1110 acts independently of its acetylation activity?

Recent research has revealed that some acetyltransferases have functions independent of their canonical enzymatic activity. To investigate this possibility for SAS1110, researchers should design experiments that distinguish between enzymatic and non-enzymatic functions:

• Catalytically inactive mutants:

  • Generate point mutations in the predicted catalytic residues of SAS1110

  • Confirm loss of acetyltransferase activity in vitro

  • Introduce these mutations in complementation constructs for in vivo studies

• Separation of function experiments:

  • Compare phenotypes between:

    • Wild-type S. aureus

    • Complete SAS1110 knockout

    • Strain expressing catalytically inactive SAS1110

  • Phenotypic rescue by catalytically inactive protein would suggest non-enzymatic functions

• Domain mapping:

  • Create truncation or domain-swap constructs

  • Test which protein regions are necessary for different functions

  • Identify domains required for protein-protein interactions vs. catalytic activity

• Mechanistic investigation:

  • Example from NAT10 research: NAT10 was found to suppress Uqcr11 and Uqcrb expression independently of its ac4C enzyme activity

  • Investigate whether SAS1110:

    • Binds directly to RNA or DNA

    • Affects localization of other proteins

    • Functions as part of larger protein complexes

• Experimental controls:

  • Include positive controls with known enzymatic functions

  • Use specific enzyme inhibitors when available

  • Measure multiple endpoints to distinguish different modes of action

This methodical approach would determine whether SAS1110 has important biological functions beyond its predicted acetyltransferase activity, similar to findings for other acetyltransferases like NAT10 .

What approaches would be most effective for identifying SAS1110 substrates in vivo?

Identifying physiologically relevant substrates for an uncharacterized acetyltransferase like SAS1110 requires multiple complementary approaches:

• Global acetylome analysis:

  • Compare protein acetylation patterns between:

    • Wild-type S. aureus

    • SAS1110 knockout strain

    • Strain overexpressing SAS1110

  • Use quantitative mass spectrometry with stable isotope labeling

  • Focus on acetylation sites showing consistent changes across conditions

• Substrate trapping approaches:

  • Generate "substrate-trapping" mutants of SAS1110 that bind but inefficiently release substrates

  • Perform pull-down experiments followed by mass spectrometry

  • Use crosslinking approaches to capture transient enzyme-substrate interactions

• Targeted metabolite analysis:

  • Focus on known acetylated metabolites in bacteria

  • Compare levels between wild-type and knockout strains

  • Use stable isotope labeling to track acetyl group transfer in vivo

• Candidate approach based on phenotypic analysis:

  • Identify metabolic pathways affected in SAS1110 knockout strains

  • Test candidate substrates from these pathways in vitro

  • Validate with in vivo approaches

• Integrative approach:

  • Combine results from multiple methods to prioritize candidates

  • Validate top candidates with:

    • In vitro enzymatic assays

    • Site-directed mutagenesis of target lysines

    • Functional studies of biological consequences

This multi-faceted strategy would help identify physiologically relevant SAS1110 substrates while minimizing false positives that can arise from single approaches.

Comparative Analysis and Evolutionary Context

How does SAS1110 compare to characterized N-acetyltransferases in other bacterial species?

Comparative analysis provides important context for understanding SAS1110's potential functions:

• Phylogenetic relationships:

  • SAS1110 belongs to the GNAT (GCN5-related N-acetyltransferase) superfamily

  • Analysis should compare SAS1110 to:

    • Other S. aureus N-acetyltransferases

    • Homologs in related Staphylococcal species

    • Functionally characterized bacterial N-acetyltransferases

• Comparison with characterized bacterial N-acetyltransferases:

• Structural and functional conservation:

  • Conserved catalytic residues across GNAT family members

  • Variability in substrate binding regions correlates with specificity

  • SAS1110-specific structural features may indicate unique functions

• Evolutionary considerations:

  • Presence/absence patterns across Staphylococcal species

  • Correlation with specific ecological niches or pathogenic potential

  • Evidence for horizontal gene transfer or adaptive evolution

• Insights from RIP and binding studies of other N-acetyltransferases:

  • NAT10 binds directly to target mRNAs and affects their localization

  • Similar mechanisms could be investigated for SAS1110

This comparative framework helps position SAS1110 within the broader context of bacterial N-acetyltransferases and generates hypotheses about its function based on evolutionary relationships.

What insights can be gained from studying SAS1110 homologs across different S. aureus strains?

Studying SAS1110 homologs across different S. aureus strains can provide valuable insights into its functional importance and strain-specific adaptations:

• Conservation analysis:

  • SAS1110 homologs have been identified across multiple S. aureus strains:

    • SAR1152 in MRSA252

    • NWMN_1086 in Newman strain

    • SAHV_1166 in other strains

    • SAV1176 in Mu50 strain

    • SAPIG1173 in other strains

  • High conservation would suggest an important core function

  • Sequence variations might indicate strain-specific adaptations

• Correlation with strain characteristics:

  • Compare SAS1110 sequence variation with:

    • Antibiotic resistance profiles

    • Host specificity

    • Virulence characteristics

    • Ecological niches

• Expression pattern differences:

  • Analyze promoter regions for regulatory differences

  • Compare expression levels across strains under standardized conditions

  • Identify strain-specific regulation that might indicate functional specialization

• Functional comparison across strains:

  • Test activity of SAS1110 homologs from different strains

  • Compare phenotypes of knockout mutants in various strain backgrounds

  • Identify strain-specific substrate preferences or activity profiles

• Experimental approaches:

  • Cross-complementation studies (expressing SAS1110 from one strain in another strain's knockout)

  • Chimeric protein studies to identify functional domains

  • Comparative genomics to identify co-evolution with other genes

This strain-comparative approach would help distinguish core functions of SAS1110 from strain-specific adaptations and potentially link sequence variations to functional differences.

How can researchers effectively design experiments to study potential regulatory mechanisms controlling SAS1110 expression?

Understanding how SAS1110 expression is regulated requires a systematic experimental approach:

• Promoter analysis and transcriptional regulation:

  • Promoter mapping:

    • Identify transcription start sites using 5' RACE

    • Create promoter-reporter fusions with varying lengths of upstream sequence

    • Identify minimal promoter region required for expression

  • Transcription factor identification:

    • Perform DNA pull-down assays with the SAS1110 promoter region

    • Use techniques like ChIP-seq to identify binding factors

    • Apply methods similar to those used to identify Hes1 as a regulator of Nat10

  • Condition-dependent regulation:

    • Test expression under varied conditions:

      • Nutrient limitation (particularly sulfur)

      • Oxidative stress

      • Antibiotic exposure

      • Growth phases

• Post-transcriptional regulation:

  • mRNA stability analysis:

    • Measure SAS1110 mRNA half-life under different conditions

    • Identify RNA-binding proteins that interact with SAS1110 transcripts

  • Small RNA regulation:

    • Identify potential sRNA regulators using computational prediction

    • Validate using reporter assays and direct binding studies

• Experimental design strategies:

  • Regulatory element mapping:

    • Create a series of promoter mutations to identify critical regulatory elements

    • Use reporter systems (e.g., luciferase, GFP) to quantify expression

    • Develop inducible systems to study dynamic regulation

  • Network analysis:

    • Identify genes co-regulated with SAS1110

    • Map the regulatory circuit controlling SAS1110 expression

    • Compare with regulation of other acetyltransferases

• Integration with global regulatory systems:

  • Examine regulation by:

    • Global stress response systems

    • Quorum sensing systems

    • Metabolic regulators (particularly sulfur metabolism regulators like CymR)

This systematic approach would elucidate the complex regulatory mechanisms controlling SAS1110 expression and provide insights into its physiological roles and integration with S. aureus regulatory networks.

Technological Approaches and Future Directions

What cutting-edge technologies could accelerate functional characterization of SAS1110?

Emerging technologies offer powerful new approaches for characterizing uncharacterized proteins like SAS1110:

• CRISPR-based technologies:

  • CRISPR interference (CRISPRi) for conditional knockdown

  • CRISPR activation (CRISPRa) for controlled overexpression

  • CRISPR scanning to identify essential domains

  • Base editing for targeted mutagenesis without double-strand breaks

• Advanced structural biology approaches:

  • Cryo-EM for structure determination without crystallization

  • AlphaFold2 and other AI-based structure prediction methods

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • Time-resolved structural biology to capture enzyme-substrate interactions

• Single-cell technologies:

  • Single-cell RNA-seq to identify cell-to-cell variation in SAS1110 expression

  • Single-cell proteomics to study protein-level heterogeneity

  • Spatial transcriptomics to map expression in infection models

• High-throughput functional screening:

  • Activity-based protein profiling with tailored probes

  • Pooled CRISPR screens to identify genetic interactions

  • Chemogenomic profiling to identify compounds that interact with SAS1110 function

• Advanced imaging techniques:

  • Super-resolution microscopy to track SAS1110 localization

  • FRET-based sensors to monitor SAS1110 activity in real-time

  • Correlative light and electron microscopy to link function to ultrastructure

These cutting-edge approaches can overcome limitations of traditional methods and provide multi-dimensional insights into SAS1110 function.

How might understanding SAS1110 contribute to broader knowledge of bacterial acetyltransferases?

Characterizing SAS1110 has potential to advance the broader field of bacterial acetyltransferase research:

• Expanding functional diversity:

  • Most characterized bacterial acetyltransferases function in:

    • Antibiotic resistance

    • Metabolic regulation

    • Protein modification

  • SAS1110 may reveal novel functions or regulatory mechanisms

• Novel substrate recognition mechanisms:

  • Understanding how SAS1110 recognizes its substrates could reveal:

    • New structural motifs for substrate recognition

    • Mechanisms for achieving substrate specificity

    • Evolution of enzyme-substrate interactions

• Non-canonical functions:

  • Recent research has identified non-enzymatic functions of acetyltransferases:

    • NAT10 regulation of mRNA localization independent of its acetyltransferase activity

    • SAS1110 could similarly have dual functions (enzymatic and non-enzymatic)

• Regulatory network integration:

  • Elucidating how SAS1110 is regulated and how it affects cellular processes would:

    • Reveal integration of acetyltransferases into bacterial regulatory networks

    • Identify new modes of acetyltransferase regulation

    • Demonstrate how acetylation interfaces with other post-translational modifications

• Evolutionary insights:

  • Comparative analysis across species could reveal:

    • Evolutionary trajectories of bacterial acetyltransferases

    • Functional diversification mechanisms

    • Acquisition of new functions through horizontal gene transfer

These broader contributions would extend the impact of SAS1110 research beyond S. aureus biology to fundamental understanding of bacterial acetyltransferases.

What experimental design approaches can effectively distinguish between multiple potential functions of SAS1110?

Uncharacterized proteins like SAS1110 may have multiple functions. Distinguishing between these requires carefully designed experiments:

• Domain dissection approach:

  • Generate truncation or point mutation constructs that selectively disable specific functions

  • Test each construct for:

    • Enzymatic activity in vitro

    • Ability to complement knockout phenotypes

    • Protein-protein interactions

    • RNA/DNA binding

  • This approach can determine which protein regions mediate different functions

• Condition-specific testing:

  • Test SAS1110 function under diverse conditions to reveal condition-specific roles:

    • Standard growth conditions

    • Nutrient limitation (particularly sulfur limitation)

    • Stress conditions (oxidative, heat, antibiotic)

    • Host-relevant environments

  • Functions relevant only in specific conditions may be missed in standard assays

• Multi-omics integration:

  • Compare wild-type and knockout strains using:

    • Transcriptomics

    • Proteomics

    • Metabolomics

    • Acetylome analysis

  • Integrate datasets to distinguish direct vs. indirect effects

• Temporal analysis:

  • Implement systems with controlled expression (inducible promoters)

  • Monitor immediate vs. delayed effects after induction/repression

  • Distinguish primary functions from secondary adaptations

• Genetic interaction mapping:

  • Conduct synthetic genetic array analysis

  • Create double mutants with genes in related pathways

  • Identify epistatic relationships that reveal functional connections

• Substrate competition assays:

  • For enzymatic functions, determine if different substrates compete

  • For binding functions, test competition between different binding partners

  • Use these data to determine if the same or different sites mediate different functions

This multi-faceted experimental design strategy enables researchers to untangle complex, multi-functional proteins and accurately characterize the full functional spectrum of SAS1110.