Recombinant Chromobacterium violaceum 4-hydroxythreonine-4-phosphate dehydrogenase (pdxA)
Product Specs
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Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized formulations have a 12-month shelf life at -20°C/-80°C.
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Target Background
4-Hydroxythreonine-4-phosphate dehydrogenase (pdxA) from Chromobacterium violaceum catalyzes the NAD(P)-dependent oxidation of 4-(phosphooxy)-L-threonine (HTP) to 2-amino-3-oxo-4-(phosphooxy)butyric acid, which spontaneously decarboxylates to form 3-amino-2-oxopropyl phosphate (AHAP).
KEGG: cvi:CV_4231
STRING: 243365.CV_4231
Q&A
Basic Research Questions
Technical Considerations
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What analytical techniques are most appropriate for characterizing recombinant C. violaceum pdxA?
A comprehensive characterization of recombinant C. violaceum pdxA should employ multiple analytical techniques:
Structural Analysis:
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Circular Dichroism (CD) spectroscopy: To assess secondary structure composition
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Differential Scanning Fluorimetry (DSF): To determine thermal stability and ligand binding
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Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): To confirm oligomeric state
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X-ray crystallography: To determine three-dimensional structure at atomic resolution
Functional Analysis:
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Isothermal Titration Calorimetry (ITC): To measure binding affinity for substrates and cofactors
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Enzyme kinetics: To determine Michaelis-Menten parameters under varying conditions
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Metal content analysis: Using ICP-MS to quantify bound metal ions
Biophysical Properties:
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Dynamic Light Scattering (DLS): To assess monodispersity and hydrodynamic radius
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Thermal shift assays: To evaluate stability in different buffer conditions
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UV-visible spectroscopy: To monitor cofactor binding
These techniques would provide comprehensive insights into the structural and functional properties of C. violaceum pdxA, facilitating comparison with pdxA enzymes from other bacterial species.
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How can gene knockout and complementation studies of pdxA in C. violaceum be effectively designed?
Genetic manipulation of C. violaceum requires careful experimental design. A recommended approach includes:
Gene Knockout:
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Design homologous recombination constructs with:
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500-1000 bp upstream and downstream of pdxA
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Selectable marker (e.g., antibiotic resistance gene)
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Consider using CRISPR-Cas9 systems for increased efficiency
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Transform C. violaceum using:
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Electroporation (optimized parameters: 2.5 kV, 200 Ω, 25 μF)
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Conjugation with E. coli donor strain (SM10 λpir)
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Select transformants on appropriate antibiotic media
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Verify knockout by:
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PCR confirmation
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Sequencing of the modified locus
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Western blotting to confirm absence of protein
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Enzymatic assays to confirm loss of function
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Complementation:
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Clone wild-type pdxA into a broad-host-range vector (e.g., pBBR1MCS or derivatives)
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Include native promoter or inducible promoter (e.g., lac or tac)
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Transform confirmed knockout strain
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Verify restoration of:
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PdxA protein expression
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Enzyme activity
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Phenotypic characteristics (motility, violacein production)
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Virulence traits in appropriate models
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This systematic approach will allow for rigorous analysis of pdxA function in C. violaceum and provide valuable insights into its role in bacterial physiology and pathogenesis.
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