Composite

Part:BBa_M36394:Design

Designed by: Dominique Dabija, Christopher Jackson, Debha Amatya   Group: Stanford BIOE44 - S11   (2011-12-06)

Spider Silk Actuator


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 109
    Illegal PstI site found at 151
    Illegal PstI site found at 510
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 109
    Illegal PstI site found at 151
    Illegal PstI site found at 510
    Illegal NotI site found at 302
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 109
    Illegal PstI site found at 151
    Illegal PstI site found at 510
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 109
    Illegal PstI site found at 151
    Illegal PstI site found at 510
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 663


Design Notes

Our overall design had a base pair limit of 3000. The point of using of Fos and Jun leucine zippers was to attempt to get the spider silk hexamers to link up head to tail via covalent bonds in the E. coli cells. Thus, we hoped to generate extremely long chains of spider silk without coding for a huge monomer in the DNA. We were not able to spin the spider silk protein out, isolate it, or analyze its structural properties. The linkers (BBa_K133132) are eight amino acids long. They were chosen because they are low in glycine. Since the spider silk hexamer contains many glycine repeats, we thought this would be ideal. They are also not too long, which ideally would prevent Fos and Jun interacting in one hexamer product. The image below shows the actuated product: [Fos-linker-Hexamer-linker-Jun] repeated as many times as possible.


Designspidersilk.jpg

Source

RBS Leader and ATG came from BioFAB (part number BD12). BD12 is a medium strength bicistronic RBS sequence, which we chose assuming we did not want a lot of spider silk being produced to kill the cell or too little to detect. Terminator comes from BioFAB (part number pFAB391). It has 99% termination efficiency. Spider silk monomer sequence was repeated six times, and the monomer AA sequence comes from Xia et al. Xia et al. designed the sequence from the natural gene N. clavipes spidroin I. The sequence was codon optimized for E. coli using. Fos-Jun leucine zipper sequences were taken from O'Shea et al. They were also codon optimized for E.coli.

References

Xia, X.-X., Z.-G. Qian, C. S. Ki, Y. H. Park, D. L. Kaplan, and S. Y. Lee. "Native-sized Recombinant Spider Silk Protein Produced in Metabolically Engineered Escherichia Coli Results in a Strong Fiber." Proceedings of the National Academy of Sciences 107.32 (2010): 14059-4063. Print.

O'Shea, E., R. Rutkowski, W. Stafford, and P. Kim. "Preferential Heterodimer Formation by Isolated Leucine Zippers from Fos and Jun." Science 245.4918 (1989): 646-48. Print.

Codon Usage table: http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/in-vitro-genetics/codon-usage.html

mFOLD: http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form2.3

Slotta, Ute, Simone Hess, Kristina Spieß, Thusnelda Stromer, Louise Serpell, and Thomas Scheibel. "Spider Silk and Amyloid Fibrils: A Structural Comparison."Macromolecular Bioscience 7.2 (2007): 183-88. Print

Rhamnex 67K Xbrane Plasmid can be found in Gene Designer, a program made by DNA 2.0