Coding

Part:BBa_K1189020:Design

Designed by: Chris Wintersinger, Denny Hoang   Group: iGEM13_Calgary   (2013-09-17)
Revision as of 07:18, 2 November 2013 by Cmwinter (Talk | contribs)

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Light chain human ferritin with his-tag and E-coil under the lacI inducible promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 230
    Illegal AgeI site found at 881
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 746

Design notes

This part was codon optimized for expression in E. coli K12.

There is evidence in the literature that ferritin nanoparticles composed entirely of heavy ferritin subunits are more soluble in E. coli, in comparison to ferritin that is entirely light subunits (Lee et al., 2002).

Variation of subunit composition in ferritin can influence the iron uptake dynamics of the nanoparticle. To more closely match the natural uptake of iron, the iGEM Calgary 2013 team built heavy/light subunit fusions, forming ferritin nanoparticles with 12 di-subunits. This method ensures a one to one proportion of heavy and light subunits in ferritin nanoparticles. Such fusions have proven expressible in E. coil (Dehal et al, 2010). Additionally, the Calgary team used this method to reduce N-termini on ferritin by half, to reduce the number of proteins fused to it and eliminate possible steric hindrance in the 3D space around ferritin.

Source

The sequence for this part was inspired by P02792 [UniParc] and generated using commercial synthesis. We synthesized BBa_K1189020 with a promoter, strong RBS, and fusion to a protein of interest in the 2013 iGEM Calgary project. This part was plasmid switched to pSB1C3 and submitted to the Parts Registry.

References

  • Dehal, P. K., Livingston, C. F., Dunn, C. G., Buick, R., Luxton, R., & Pritchard, D. J. (2010). Magnetizable antibody‐like proteins. Biotechnology journal, 5(6), 596-604.
  • Lee, J., Kim, S. W., Kim, Y. H., & Ahn, J. Y. (2002). Active human ferritin H/L-hybrid and sequence effect on folding efficiency in< i> Escherichia coli. Biochemical and biophysical research communications, 298(2), 225-229.