DNA
O5

Part:BBa_K2054005:Design

Designed by: LAI HEI WAI   Group: iGEM16_Hong_Kong_HKU   (2016-10-14)
Revision as of 12:51, 26 October 2016 by Waiturtle (Talk | contribs) (→‎Design Notes)


Oligo 5 of DNA Tetrahedral Nanostructure


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
    COMPATIBLE WITH RFC[1000]


Design Notes

This device was modified from a recent publication about in vivo synthesis and self-assembly of DNA nanostructures. We made use of the technique described in the article to synthesize oligos for our tetrahedral nanostructure, where the sequences are generated by a software called Tiamat. This device will enable the single-stranded DNA oligo 5 to be synthesized inside cells. The ssDNA oligos produced can be purified and used to form our tetrahedral nanostructure, which consists of 5 oligos.


Below show the paper fold of our tetrahedral nanostructure and the sequence of the oligos.


Construction and production of ssDNA The design is based on the literature mentioned and contains:

a strong promoter BBa_J23100 from the Registry of standard biobricks a ‘r_oligo’ region that contains the sequence of our desired oligos and more (see below); a terminator BBa_B0054, which is also from the Registry;


The ‘r_oligo’ region will transcribe a product that contains a non-coding RNA (ncRNA) and a HIV-Terminator-Binding Site (HTBS) that exhibit a 3’-hairpin structure:

The HTBS serves as a terminator in this gene, where the HIV reverse transcriptase binds. During the reverse transcription, the binding of HIVRT initiates the elongation, which is aided by another RT murine leukemia reverse transcriptase (MLRT). RNase H then cleaves specifically the ncRNA-DNA linkages, which leaves the desired ssDNA to hang, but still attached to the HTBS on its 5’ end. RNase A then breaks to release the desired ssDNA. The following diagram (fig 2a extracted from the article) summarizes the in vivo conversion.

Source

Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7.

References