Difference between revisions of "Part:BBa K2054000:Design"

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(→‎Design Notes)
 
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The design is based on the literature and that of basic parts mentioned and contains 4 more replicates joint together by the XbaI RE site of the suffix and the SpeI RE site of the prefix.  
 
The design is based on the literature and that of basic parts mentioned and contains 4 more replicates joint together by the XbaI RE site of the suffix and the SpeI RE site of the prefix.  
  
 +
Below shows our tetrahedral nanostructure and the sequence of the oligos.
 +
 +
[[File:Tetra3d1.png|500px]]
 +
 +
One of the key features of our tetrahedral nanostructure is the strand displacement induced by the input strand. The schematic diagram below illustrates how.
 +
 +
[[File:Sdschematic.png|500px]]
 +
 +
NB: for simplicity, only the active component (i.e. O1+O5 beacon) is drawn.
 +
 +
Below is a 12% polyacrlamide gel to show the assembly (lanes 2 to 7) and the strand displacement (lanes 6, 8-10) of the tetrahedron. Lanes 2, 3, 4, 5 and 6 correspond to the individual single-stranded oligos 1, 2, 3, 4 and 5, and lane 6 has all the five oligos put together in the thermocycler, where the tetrahedron is expected. Lanes 6, 8 and 9 contains oligo 5, input strand and the dimer of O5 and input, the expected displaced product. Finally, lane 10 shows the addition of input strand to the tetrahedron, inducing the strand displacement. As expected, this lane contains the displaced output product, of the same size and the output marker in lane 9.
 +
 +
[[File:Sdtetra1.png|500px]] 
 +
 +
Construction and production of ssDNA
 +
The design is based on the literature mentioned and contains:
 +
 +
[[File:plasmidconstruct1.png|500px]]
 +
 +
a strong promoter BBa_J23100 from the Registry of standard biobricks;
  
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Each replicate contains the following:
 
−
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 ‘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;
 
a terminator BBa_B0054, which is also from the Registry;
  
  
−
Each ‘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 ‘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.  
+
[[File:plasmidconstruct2.png|500px]]
  
 +
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 summarizes the in vivo conversion.
  
−
The following diagram (fig 2a extracted from the article) summarizes the in vivo conversion.
+
[[File:plasmidconstruct3.png|800px]]
  
 
===Source===
 
===Source===

Latest revision as of 03:00, 30 October 2016


All Oligos for Tetrahedral Nanostructure


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal NheI site found at 29
    Illegal NheI site found at 52
    Illegal NheI site found at 355
    Illegal NheI site found at 378
    Illegal NheI site found at 651
    Illegal NheI site found at 674
    Illegal NheI site found at 964
    Illegal NotI site found at 7
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This is the fusion product of our basic parts BBa_K2054001 – BBa_K2054005. This device will enable all the single-stranded DNA oligos (total 5) to be synthesized inside cells, and to achieve the self-assembly of our desired nanostructure.


Construction and production of ssDNA The design is based on the literature and that of basic parts mentioned and contains 4 more replicates joint together by the XbaI RE site of the suffix and the SpeI RE site of the prefix.

Below shows our tetrahedral nanostructure and the sequence of the oligos.

Tetra3d1.png

One of the key features of our tetrahedral nanostructure is the strand displacement induced by the input strand. The schematic diagram below illustrates how.

Sdschematic.png

NB: for simplicity, only the active component (i.e. O1+O5 beacon) is drawn.

Below is a 12% polyacrlamide gel to show the assembly (lanes 2 to 7) and the strand displacement (lanes 6, 8-10) of the tetrahedron. Lanes 2, 3, 4, 5 and 6 correspond to the individual single-stranded oligos 1, 2, 3, 4 and 5, and lane 6 has all the five oligos put together in the thermocycler, where the tetrahedron is expected. Lanes 6, 8 and 9 contains oligo 5, input strand and the dimer of O5 and input, the expected displaced product. Finally, lane 10 shows the addition of input strand to the tetrahedron, inducing the strand displacement. As expected, this lane contains the displaced output product, of the same size and the output marker in lane 9.

Sdtetra1.png

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

Plasmidconstruct1.png

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:

Plasmidconstruct2.png

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 summarizes the in vivo conversion.

Plasmidconstruct3.png

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

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