Part:BBa_K2916059:Design

Toehold for Bois Noir 2.1

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 AgeI site found at 96
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

1. According to the paper (Green et al., 2014), our prototype design has a loop of 15nts at the head of the hairpin. Based on iGEM RBS registry, we chose the RBS from the community collection, which is the same used in the biobits paper. Also suggested by Vimberg et al., 2007 (a A/U RICH regions in the pre-RBS will enhance the the rate of translation). So in our design we have the following sequence as the pre-RBS which has a A/U content of 57%: gacaugu 2. According to iGEM registry, the optimal distance between the starting codon and RBS is around 6-7 nts, in our first design we choose 6 nts(also A/U enriched): cuuuaa 3. For starting codon it will always be "aug", refering to Green et al., 2014, it is sequestered by 3 non-complementory neuclotides: cca 4. Therefore, we have the upper part of our hairpin sequence and structure: cca cuuuaa gacauguagaggaga uuaaag aug (NED:8.8%) normalized ensemble defect using NUPACK 5. Then, we also tested a functioning toehold on the Biobits paper (pCOLA banana sensor sfGFP). Their common haipin sequence is: gga cttta gaac agaggaga taaag atg

6. Combining both we have the upper part of our hairpin sequence and structure: cca cuuuaa gaacagaggaga uuaaag aug (NED: 11.8%) normalized ensemble defect using NUPACK

For the toehold folding part: Using the equation from Green et al. 2014, our toehold switch is sorted by its Design score from lowest to highest. From their function and way of normalising, the lower the score the better.

“Where Φ(i) is the design score for the sensor at location i of the mRNA, lmRNA is the local single-strandedness of the mRNA at the sensor binding site, ltoehold is the local single-strandedness of the toehold of the sensor, and nsensor is the normalised ensemble defect of the sensor. The score ‘-‘ above certain variables indicates they were normalised as shown in this equation: x = x/max(x). the score weight factors used were β1 = 5, β2 = 4 and β3 = 3.” ——Green et al., 2014

Source

Order toehold as primers and perform PCR on the repressed gene plasmid.

References

Green, Alexander A., et al. “Toehold Switches: De-Novo-Designed Regulators of Gene Expression.” Cell, vol. 159, no. 4, Nov. 2014, pp. 925–39. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.002.