RNA

Part:BBa_K3453024

Designed by: Maeva Cherriere   Group: iGEM20_Evry_Paris-Saclay   (2020-10-04)


DmMatK Trigger 2.1

This part is a trigger for sequence-based detection of rosewood. It contains a fragment of the MatK gene of Dalbergia maritima var. pubescens (BOLD Acc. n° MADA155-14, MADA063-14, MADA062-14, MADA158-14, MADA065-14, MADA104-14, MADA023-14, MADA071-14, MADA110-14) [1].

Usage and Biology

A toehold switch is an RNA–based device containing a ribosome binding site (RBS) and an ATG start codon embedded in a hairpin structure that blocks translation initiation [2]. The hairpin can be unfolded upon binding of a trigger RNA thereby exposing the RBS and the ATG start codon permitting translation of the reporter protein (Figure 1).


T--Evry Paris-Saclay--toehold-switch.png

Figure 1. Toehold switches principle.

This part is a trigger for the Rosewood DmMatK Toehold Switch 2.1 (BBa_K3453014) and was designed using our internal toehold switch pipeline which calls NUPACK [3] and RBS Calculator for switch prediction and ranking [4].

The functionality of this part was tested under controlled of the T7 promoter (BBa_K2150031) and the strong SBa_000587 synthetic terminator (BBa_K3453000) in the composite part BBa_K3453124.

This part proved to be functional: the transcribed rosewood RNA was able to bind to the corresponding toehold switch and a readable output was generated.

Full results are available on the BBa_K3453114 page in the registry.

References

[1] Hassold S, Lowry PP 2nd, Bauert MR, Razafintsalama A, Ramamonjisoa L, Widmer A. DNA barcoding of Malagasy rosewoods: towards a molecular identification of CITES-listed Dalbergia species. PLoS One (2016) 11, e0157881.

[2] Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell (2014) 159, 925-939.

[3] Zadeh JN, Steenberg CD, Bois JS, Wolfe BR, Pierce MB, Khan AR, Dirks RM, Pierce NA. NUPACK: Analysis and design of nucleic acid systems. Journal of Computational Chemistry (2011) 32, 170–173.

[4] Salis HM. The ribosome binding site calculator. Methods in Enzymology (2011) 498, 19–42.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 26
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 26
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 26
    Illegal BamHI site found at 37
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 26
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 26
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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