Part:BBa_K3453035

Rosewood DmRbcL Toehold Switch 2.2

This part is a toehold switch sensor for sequence-based detection of rosewood. It targets a fragment of the RbcL gene of Dalbergia maritima var. pubescens (BBa_K3453040).

Usage and Biology

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

Figure 1. Toehold switches principle.

This part is a toehold switch sensor that targets a fragment of the RbcL gene of Dalbergia maritima var. pubescens (BBa_K3453040). It was designed using our internal toehold switch pipeline which calls NUPACK [2] and RBS Calculator for switch prediction and ranking [3]. Its secondary structure predicted by NUPACK web server [2] using default parameters is represented in Figure 2 and here-after in dot-bracket notation.

.....((......))((((((((((((((((((...............)))))))))))))))))).............((((.....))))....

Figure 2. Secondary-structure prediction of this part with the ATG of the reporter gene. The prediction was realised using the NUPACK web server [2] with default parameters and graphically represented using the forna RNA secondary structure visualization tool [4]. Nucleotides were coloured to match the different segments in Figure 1.

The corresponding trigger sequence of this toehold switch is BBa_K3453045.

The functionality of this part was tested using sfGFP-LVAtag (BBa_K2675006) as a reporter. The expression was controlled by the T7 promoter (BBa_K2150031) and the strong SBa_000587 synthetic terminator (BBa_K3453000) in the composite part BBa_K3453135.

This part proved to be functional: a readable output was generated only in the presence of rosewood RNA.

Full results are available on the BBa_K3453135 page in the registry.

References

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

[2] 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.

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

[4] Kerpedjiev P, Hammer S, Hofacker IL. Forna (force-directed RNA): Simple and effective online RNA secondary structure diagrams. Bioinformatics (Oxford, England) (2015) 31, 3377–3379.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]