Coding
lFTN

Part:BBa_K1189024:Design

Designed by: Chris Wintersinger, Denny Hoang   Group: iGEM13_Calgary   (2013-09-17)
Revision as of 01:56, 21 October 2020 by Jlhsiao (Talk | contribs)

Light chain human ferritin


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 391

Design notes

This part was codon optimized for expression in E. coli K12. Note that it contains a BsaI cut site which should be removed in order to apply it to Golden Gate assembly methods.

There is evidence in the literature that ferritin nanoparticles composed entirely of light ferritin subunits are less soluble in E. coli, in comparison to ferritin that is entirely heavy subunits or a mixture of the two (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 disubunits. 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. The light ferritin subunit was biobricked from this construct using PCR and isothermal assembly.

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.

    • Contribution (Waterloo iGEM)

    Boyd, D., Vecoli, C., Belcher, D.M., Jain, S.W., & Drysdale, J.W. (1985). Structural and Functional Relationships of Human Ferritin H and L Chains Deduced from cDNA Clones. The Journal of Biological Chemistry, 260(21). 11755-11761.

    Levi, S. et al. (1988). Mechanism of Ferritin Iron Uptake: Activity of the H-chain and Deletion Mapping of the Ferro-oxidase Site. The Journal of Biological Activity, 263(34). 18086-18092.

    Santambrogio, P. et al. (1993). Production and Characterization of Recombinant Heteropolymers of Human Ferritin H and L Chains. The Journal of Biological Chemistry, 268(17). 12744-12748.