Coding
spilk 7.0.

Part:BBa_M36500:Design

Designed by: Dominique Dabija, Christopher Jackson, Debha Amatya   Group: Stanford BIOE44 - S11   (2011-12-03)

Spider Silk Hexamer


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 254
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 254
    Illegal NotI site found at 46
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 254
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 254
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 407


Design Notes

The design of this hexamer sequence was rather lengthy and complicated. As a basis, we took the monomer AA acid sequence from Xia et. al: SGRGGLGGQGAGMAAAAAMGGAGQGGYGGLGSQGT. We then multiplied this sequence out to a hexamer and selected optimal codons for E. coli, using a codon usage table specified in the references section. The DNA sequence of the 6 monomers was not the same: we also needed to minimize the number of stem loops in the mRNA structure if we ever wanted protein produced, so we used a method of trial and error to design a varying sequence, test it on mFOLD, and redesign until getting the best one. Originally, we had designed a 12-mer. Our team was given a limit of 3,000 base pairs for a total design (this part is only a piece of that design) but the 12-mer would have fit. However, the 12-mer had too many repeats over 10 base pairs and would have been too difficult to synthesize for DNA 2.0, the company we sent our design to. Thus we had to cut it down to a hexamer.

It is also interesting to note that Xia et al made a 96-mer of the sequence above and had to up regulate the glycyl-tRNA pool in their cells. This would have been impossible given our design considerations, and we wanted to try something else anyway.

Source

Xia et al. modified the sequence from N. clavipes Spidroin I.

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.

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