Coding

Part:BBa_K3853009

Designed by: Peng Luo   Group: iGEM21_CPU_CHINA   (2021-09-19)
Revision as of 04:45, 21 October 2021 by Lp-tiffany (Talk | contribs)


his-tag-SpyTag-AAO

Aryl alcohol oxidase (AAO) is an enzyme containing flavin-adenine-dinucleotide (FAD). This is the version with SpyTag. In order to constructed our multi-enzyme complex, we introduced the SpyTag/SpyCatcher system. We added SpyTag to the N-terminus of each protein through 10 × ELP, which is a oligopeptide linker that does not affect the function of the protein it attaches to, as described in the literature[1]. So this part can be combined with dCas9-SpyCatcher through covalent isopeptide action, thereby being immobilized on dsDNA. His-tag was added to purify the protein. We use BBa_K3853053 to construct the expression system to express and purify the protein.

Biology

Aryl alcohol oxidase (AAO, EC 1.1.3.7), a member of the glucose-methanol-choline oxidase / dehydrogenase (GMC) superfamily, is an enzyme containing flavin-adenine-dinucleotide (FAD) that catalyze the oxidation of aromatic and aliphatic allylic primary alcohols to the corresponding aldehydes while reducing molecular oxygen to H2O2(The mechanism has shown in Fig. 1).

Usage

References

[1] Lim, S., Kim, J., Kim, Y., Xu, D. & Clark, D. S. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chemical communications (Cambridge, England) 56, 4950-4953, doi:10.1039/d0cc01174f (2020).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 699
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 412
    Illegal BamHI site found at 1411
    Illegal BamHI site found at 1798
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None