Recombinant Synechococcus sp. UPF0176 protein SYNW0932 (SYNW0932)

What are the optimal storage conditions for maintaining SYNW0932 stability?

The stability of recombinant SYNW0932 is influenced by storage conditions, buffer composition, and protein formulation. For optimal stability:

• Lyophilized form: Store at -20°C/-80°C for up to 12 months

• Liquid form: Store at -20°C/-80°C for up to 6 months

When reconstituting the lyophilized protein:

• Centrifuge the vial briefly before opening

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot to avoid repeated freeze-thaw cycles

• For working aliquots, store at 4°C for up to one week

Repeated freeze-thaw cycles significantly reduce protein stability and should be avoided to maintain functional integrity.

How should I design experiments to identify the function of SYNW0932?

When designing experiments to elucidate SYNW0932 function, a multi-faceted approach is recommended:

• Gene knockout studies:

  • Create markerless knockout strains using techniques similar to those developed for other Synechococcus species

  • Use the PheS-based counter selection method which has proven effective in cyanobacteria

  • Analyze phenotypic changes under various growth conditions

• Expression profiling:

  • Monitor SYNW0932 expression under different environmental conditions (light intensity, temperature, nutrient availability)

  • Compare expression patterns with proteins of known function

• Protein interaction studies:

  • Perform pull-down assays using recombinant SYNW0932 as bait

  • Use yeast two-hybrid or bacterial two-hybrid systems to identify interaction partners

• Comparative genomics:

  • Analyze homologs in other cyanobacterial species

  • Look for conserved genomic contexts that might suggest functional relationships

• Structural analysis:

  • Determine the three-dimensional structure through X-ray crystallography or cryo-EM

  • Identify potential active sites or binding domains

Document all experimental conditions precisely, include appropriate controls, and validate findings using multiple independent approaches.

What growth parameters affect Synechococcus biomass production and protein expression?

Growth parameters significantly impact both Synechococcus biomass accumulation and recombinant protein expression. Key parameters include:

For Synechococcus sp. UTEX 2973, biomass can increase from 0.13±0.01 mg/ml to 0.87±0.03 mg/ml in just 16 hours under optimal conditions, demonstrating the rapid growth potential of optimized Synechococcus strains . Both high light intensity and CO₂ supplementation are critical factors supporting rapid growth and protein production.

How can I establish a markerless genetic manipulation system for studying SYNW0932?

For advanced genetic manipulation of SYNW0932 in Synechococcus, a markerless approach offers significant advantages. Here's a methodological framework:

• PheS-based counter selection system:

  • Introduce a mutated phenylalanyl-tRNA synthetase gene (pheS) containing the T261A and A303G substitutions

  • This creates susceptibility to the phenylalanine analog p-chlorophenylalanine (PCPA)

  • The optimal PCPA concentration for selection is 15-20 μg/mL

• Two-step recombination protocol:

  • First transformation: Introduce a construct containing the mutated pheS gene and an antibiotic resistance marker into the target locus

  • Selection: Select transformants on media with the appropriate antibiotic

  • Second transformation: Transform with a construct containing the desired modification

  • Counter-selection: Select on media containing PCPA to isolate markerless recombinants

• Verification of recombinants:

  • PCR analysis to confirm complete segregation

  • Sequencing to verify the desired modification

  • Phenotypic analysis to ensure no off-target effects

This approach has been successfully demonstrated for creating double markerless knockin recombinants in Synechococcus, indicating its utility for multiple successive genetic manipulations .

What considerations are important when using recombinant antibodies to study SYNW0932?

When using recombinant antibodies to study SYNW0932, several methodological considerations are critical:

• Antibody format selection:

  • Full IgG: Provides bivalent binding and Fc-mediated effector functions

  • Fab fragments: Smaller size, better tissue penetration, no Fc effector functions

  • ScFv: Even smaller size, potentially reduced immunogenicity

  • Consider the experimental requirements when selecting format

• Engineering considerations:

  • Add epitope tags (His, FLAG, Halo) for detection or purification

  • Incorporate fluorescent protein fusions for localization studies

  • Engineer mutations to enhance binding affinity or specificity

• Expression and purification:

  • Use dual promoter plasmids for efficient co-expression of heavy and light chains

  • Consider yeast expression systems for complex proteins

  • Purify using affinity chromatography methods appropriate for the antibody format

• Validation protocols:

  • Verify binding specificity through Western blot, immunoprecipitation

  • Determine binding affinity using surface plasmon resonance

  • Test for cross-reactivity with related proteins

  • Validate in the intended experimental application

• Storage and handling:

  • Store according to stability profile (typically at -20°C/-80°C)

  • Avoid repeated freeze-thaw cycles

  • Prepare working aliquots for short-term use

Recombinant antibodies offer advantages over conventional antibodies, including unambiguous identification through DNA sequencing, reliable expression, and opportunities for engineering to enhance utility .

How can I apply CRISPR-Cas technologies to study SYNW0932 function in Synechococcus?

CRISPR-Cas technologies provide powerful tools for studying SYNW0932 function in Synechococcus. Here's a methodological framework:

• CRISPR interference (CRISPRi) for gene knockdown:

  • Design a DAPG-inducible dCas9-based CRISPRi system targeting SYNW0932

  • Select appropriate sgRNA targeting sequences using tools optimized for cyanobacteria

  • Transform with a plasmid containing the dCas9 and sgRNA expression cassettes

  • Induce with DAPG to achieve controlled repression

• CRISPR-Cas12a for precise genome editing:

  • Design a modular method for generating markerless mutants

  • Target single insertion with expected efficiency of 31-81%

  • For multiplex double insertion, expect efficiency around 25%

• Implementation strategy:

  • Characterize neutral genomic integration sites that don't affect growth

  • Test a suite of constitutive promoters and terminators for optimal expression

  • Implement the 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system which shows tight regulation with a 228-fold dynamic range

• Analytical approaches:

  • Measure transcript levels using RT-qPCR

  • Analyze protein levels via Western blotting

  • Assess phenotypic changes under various conditions

  • Perform comparative -omics analyses (transcriptomics, proteomics, metabolomics)

This approach leverages recent advances in cyanobacterial genetic tools, specifically those developed for Synechococcus strains with high engineering potential .

What experimental design principles should be applied when studying SYNW0932 function?

When designing experiments to investigate SYNW0932 function, apply these rigorous experimental design principles:

• Define clear variables:

  • Independent variable (IV): The factor you manipulate (e.g., SYNW0932 expression levels)

  • Dependent variable (DV): The measured outcome (e.g., growth rate, metabolite production)

  • Controlled variables (CV): Factors kept constant (e.g., temperature, light intensity, media composition)

  • Experimental control: Typically wild-type strain or empty vector control

• Establish a robust experimental protocol:

  • Include at least three replicates for each condition

  • Randomize the order of experiments to minimize systematic errors

  • Include appropriate positive and negative controls

  • Create detailed set-up diagrams for reproducibility

• Advanced statistical considerations:

  • For Division C level analysis, include experimental controls, significant figures, and abstract

  • Apply chi-squared analysis for assessing differences between experimental groups

  • Consider accuracy vs. precision in measurements

• Address potential experimental errors:

  • Identify possible sources of error in experimental setup

  • Document human errors in data collection or execution

  • Describe how these errors may affect results (direction and magnitude)

• Applications and future directions:

  • Propose three variations to improve experiment accuracy

  • Suggest alternative approaches to test the hypothesis

  • Recommend future experiments related to the dependent variable

This structured approach ensures rigorous scientific investigation and facilitates reproducibility of results across different research groups.

How can I predict and optimize the recombinant expression of SYNW0932 in different host systems?

Predicting and optimizing recombinant expression of SYNW0932 requires consideration of multiple factors across different expression systems:

• Computational prediction approaches:

  • Utilize deep learning models like CysPresso that leverage protein representations from AlphaFold2

  • These models can predict recombinant protein expression with AUC values of 0.798-0.852

  • Apply time series classification methods with random convolutional kernels for enhanced prediction accuracy

• Host system optimization:

  Host System	Advantages	Optimization Strategies
  E. coli	Rapid growth, high yields	Codon optimization, fusion tags, chaperone co-expression
  Yeast	Post-translational modifications	Signal sequence optimization, strain selection
  Mammalian cells	Complex protein folding	Vector design, cell line selection, transfection optimization
  Cyanobacteria	Native environment	Light intensity, CO₂ concentration, temperature regulation

• Expression vector design considerations:

  • For cyanobacterial expression, the CyanoGate MoClo system provides modular parts

  • Test multiple constitutive promoters, terminators, and inducible systems

  • Consider the DAPG-inducible PhlF repressor system with its 228-fold dynamic range

  • For mammalian expression, evaluate CysPresso predictions to identify expressible constructs

• Key parameters for optimization:

  • Codon usage harmonization with host system

  • mRNA secondary structure at translation initiation site

  • Protein solubility and potential toxicity to host

  • Induction conditions (timing, temperature, inducer concentration)

  • Harvest time optimization

By combining computational prediction with systematic optimization of expression conditions, researchers can significantly improve SYNW0932 production across different host systems.

What approaches should be used to investigate potential recombination between SYNW0932 and other genes in Synechococcus communities?

Investigating recombination events involving SYNW0932 requires sophisticated methodological approaches:

• Detection of recombination events:

  • Identify single nucleotide polymorphisms (SNPs) that are phylogenetically informative

  • Screen for unusual combinations of SNPs that suggest recombination

  • Apply lightweight computational approaches suitable for screening large sequence databases

  • Assess assembly quality of potentially recombinant genomes through read mapping and pileup analysis

• Reverse genetics approach for validation:

  • Develop a bacterial artificial chromosome (BAC)-based system for stable maintenance of full-length cDNA clones

  • Generate recombinant strains with predicted recombination events

  • Assess phenotypic consequences of these recombination events

  • Evaluate fitness impacts through competition assays

• NIH Guidelines compliance:

  • Follow established guidelines for research involving recombinant or synthetic nucleic acid molecules

  • Consider that recombinant nucleic acids are defined as "molecules that a) are constructed by joining nucleic acid molecules and b) can replicate in a living cell"

  • Ensure proper institutional biosafety committee approval for studies

• Analytical framework:

  • Determine rates of recombination through phylogenetic analysis

  • Identify potential recombination hotspots in the genome

  • Assess the co-circulation of predicted parent clades in natural environments

  • Estimate the percentage of circulating viruses that are recombinant (adjusting for detection biases)

This multifaceted approach enables rigorous investigation of recombination events involving SYNW0932 while ensuring compliance with regulatory requirements.