Composite
rAIP

Part:BBa_K2027000

Designed by: Charles Gleason, Michael Becich   Group: iGEM16_Stanford-Brown   (2016-09-07)
Revision as of 10:24, 24 October 2016 by Htale1 (Talk | contribs) (→‎Characterization)


Recombinant Apoptosis-Inducing Protein (L-lysine Alpha Oxidase)

This part is a native Scomber japonicus enzyme codon-optimized for Escherichia coli. The conjugated tag should allow for purification and visualization with anti-FLAG antibodies, visualization with Lumio™ Green, and purification with nickel columns. Tani et al. characterized this enzyme in their quest for synthesis of L-pipecolic acid from racemic lysine after nickel column purification, and we verified the function of our part in a similar way. More information can be found on the experience page for this part.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1004
    Illegal BglII site found at 1032
    Illegal BamHI site found at 767
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 25


Characterization

We were able to transform this part into E. coli and induce protein synthesis using IPTG. We extracted this protein using Ni-NTA resin columns and purified it successfully.


Using the bicinchoninic acid assay (BCA) to determine the total concentration of protein in our final elution buffer, we found that our purified protein had a concentration of 149.317 mg/mL (shown below from Nanodrop 2000 software).

T--Stanford-Brown--raip_BCA_ss.png

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Parameters
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