Part:BBa_K3453076

Grapevine VvTrnL-UAA Toehold Switch 1.1.3

This part is a toehold switch sensor for sequence-based detection of grapevine. It targets a fragment of the TrnL-UAA gene of Vitis vinifera in reverse orientation (BBa_K3453084).

It is a technical improvement of the Grapevine VvTrnL-UAA Toehold Switch 1.1 BBa_K2916050.

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 TrnL-UAA gene of Vitis vinifera in reverse orientation (BBa_K3453084).

It's 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 [3]. Nucleotides were coloured to match the different segments in Figure 1.

This part is a technical improvement of the Grapevine VvTrnL-UAA Toehold Switch 1.1 (BBa_K2916050). Indeed, RBS strength may be may be responsible for the low sfGFP expression observed from all BBa_K2916050 (T7 promoter + grapevine switch 1.1) derived parts that we analysed (BBa_K2916068, BBa_K3453170, BBa_K3453171, BBa_K3453172). To investigate this possibility we modified the RBS stem-loop region of BBa_K2916050 and transformed it into the ‘standard’ RBS stem-loop region (BBa_K3453005) of the Series B of toehold switch sensors for Zika virus detection [4] and of the BioBits™ toeholds [5] that was used in the positive control BBa_K3453105. The resulting part (BBa_K3453076) was denoted the Grapevine Switch 1.1.3. A truncated version lacking the trigger binding site (BBa_K3453078) was also designed to mimic the open (unfolded) state of this toehold switch.

The functionality of this part was tested using either sfGFP (BBa_I746916) or sfGFP-LVAtag (BBa_K2675006) as reporters. The expression was controlled by the T7 promoter (BBa_K2150031) and either the BBa_B0015 or the strong SBa_000587 synthetic terminator (BBa_K3453000) in the composite parts BBa_K3453176 and BBa_K3453177 respectively.

This part proved to be functional: a readable output was generated only in the presence of grapevine RNA artificially expressed in E. coli in reverse orientation.

Full results are available on the BBa_K3453176 and BBa_K3453177 pages 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] 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.

[4] Pardee K, Green AA, Takahashi MK, Braff D, Lambert G, Lee JW, Ferrante T, Ma D, Donghia N, Fan M, Daringer NM, Bosch I, Dudley DM, O'Connor DH, Gehrke L, Collins JJ. Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell (2016) 165, 1255-1266.

[5] Huang A, Nguyen PQ, Stark JC, Takahashi MK, Donghia N, Ferrante T, Dy AJ, Hsu KJ, Dubner RS, Pardee K, Jewett MC, Collins JJ. BioBits™ Explorer: A modular synthetic biology education kit. Sci Adv (2018) 4, eaat5105.

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]