Composite

Part:BBa_K2974316:Design

Designed by: Janet Standeven, Megan Hong, Courtney Taing, Julia Chang, Shanthi Hegde, Sri Pinnamareddy   Group: iGEM19_Lambert_GA   (2019-10-16)


Medium Promoter (BBa_J23106) C. elegans Toehold GFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 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 688


Design Notes

By using the lin-4 gene pre-mRNA primary transcript F59G1.6 sequence found on WormBank, we designed our toehold switch RNA sequence using NUPACK software and software code provided by Yan Zhang. In addition to gaining the toehold switch sequence, we also obtained a complementary RNA trigger sequence. By inputting the minimum free energy structures for each of the switch and trigger combinations, NUPACK showed the possible structure, allowing us to determine whether or not each switch and trigger would have a high probability of working. The minimum free energy shown below demonstrates the strength of repression for the switch RNA and the single-strandedness of the trigger RNA for the activated complex. A negative ∆GRBS-linker value is correlated to a lower switch dynamic range.

T--Lambert GA--ToeholdMegan2.png

Figure 1: Calculated optimal toehold switch structure design


Source

The lin-4 gene pre-mRNA primary transcript F59G1.6 (non-toxic) is native to the genome of C. elegans.

References