Part:BBa_K4432002
PANTR1 Toehold Switch Sensor 02
This part is a toehold switch sensor for sequence-based detection of PANTR1, a long non coding (lnc) RNA overexpressed in different types of cancer cells.
Usage and Biology
A toehold switch [1] (Figure 1) is an RNA-based device is composed of several parts which are essential for the efficient functioning of our biosensor :
- a Trigger Binding Site (TBS) which is a sequence complementary to the RNA target to be detected
- a Ribosome Binding Site (RBS) which allows the start of the translation only when the toehold switch is unfolded
- a start codon AUG which constitutes the start of the translation
- a linker which constitutes a bridge between the Start codon and our gene of interest
- a repressed gene of interest that is translated into a protein only when the hairpin is unfolded and the RBS exposed
Figure 1. Principle of the toehold switch (inspired from iGEM Evry Paris-Saclay 2020).
The functioning comes as follows :
The binding between the RNA biomarker and the toehold switch unfolds the hairpin structure. The RBS becomes accessible now so that the translation can take place, and the initially repressed gene of interest can then be translated into a protein.
This part is a toehold switch sensor that targets a fragment of the PANTR1 lncRNA.
It was designed using the web tool developed by To et al. [2] and follows the architecture of the Series B of toehold switch sensors for Zika virus detection [3] and of the BioBits™ toeholds [4]. Its secondary predicted structure 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 web tool developed by To et al. [2] and graphically represented using the forna RNA secondary structure visualization tool [5]. Nucleotides were coloured to match the different segments in Figure 1.
The corresponding trigger sequence of this toehold switch is BBa_K4432202.
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_K4432102.
This part proved not to be functional: no fluorescence is observed in any condition. The sfGFP expression is tightly controlled and the presence of the trigger does not allow the hairpin to unfold.
Full results are available on the BBa_K4432102 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] To AC, Chu DH, Wang AR, Li FC, Chiu AW, Gao DY, Choi CHJ, Kong SK, Chan TF, Chan KM, Yip KY. A comprehensive web tool for toehold switch design. Bioinformatics (2018) 34, 2862-2864.
[3] 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.
[4] 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.
[5] 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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