Part:BBa_K5243001:Design
Expression vector for oxalate oxidase
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 282
Illegal BglII site found at 529
Illegal BglII site found at 1198
Illegal BglII site found at 1347
Illegal BamHI site found at 65
Illegal XhoI site found at 1315 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2955
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 2937
Illegal SapI.rc site found at 1429
Design Notes
Codon optimization: The oxalate oxidase gene was codon-optimized for E. coli to ensure efficient expression and reduce translational pausing. Inducibility: The araBAD promoter was selected for its tight regulation, allowing expression only in the presence of arabinose, which minimizes metabolic burden on the host until induction is necessary. Purification efficiency: The addition of a GST tag enables simple and efficient purification using glutathione affinity chromatography, while the TEV cleavage site allows for removal of the tag after purification to yield a native form of the enzyme. Toxic byproducts: The generation of hydrogen peroxide as a byproduct of oxalate oxidation was considered, and care was taken to ensure the E. coli host would tolerate this oxidative stress during expression."
Source
This part is derived from the wheat oxalate oxidase gene, which has been codon-optimized for expression in E. coli. The araBAD promoter and regulatory elements were adapted from the Escherichia coli arabinose operon, ensuring compatibility with bacterial expression systems. The TEV cleavage site and GST tag were sourced from standard expression vectors commonly used for protein purification.