Difference between revisions of "Part:BBa K3453034"

 
 
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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'' ([[Part:BBa_K3453040|BBa_K3453040]]).
  
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===Usage and Biology===
 
===Usage and Biology===
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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).
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[[File: T--Evry_Paris-Saclay--toehold-switch.png|600px]]
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Figure 1. Toehold switches principle.
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This part is a toehold switch sensor that targets a fragment of the RbcL gene of ''Dalbergia maritima'' var. ''pubescens'' ([[Part:BBa_K3453040|BBa_K3453040]]).
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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.
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...............((((((((((..((((((...............))))))..))))))))))..((....))..(((((.....)))))...
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[[File: T--Evry_Paris-Saclay--RNAstructure_p70_DmRbcL2-1.png|center]]
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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.
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The corresponding trigger sequence of this toehold switch is [[Part:BBa_K3453044|BBa_K3453044]].
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The functionality of this part was tested using sfGFP-LVAtag ([[Part:BBa_K2675006|BBa_K2675006]]) as a reporter. The expression was controlled by the T7 promoter ([[Part:BBa_K2150031|BBa_K2150031]]) and the strong SBa_000587 synthetic terminator ([[Part:BBa_K3453000|BBa_K3453000]]) in the composite part [[Part:BBa_K3453134|BBa_K3453134]].
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This part proved to be functional: a readable output was generated only in the presence of rosewood RNA.
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Full results are available on the [[Part:BBa_K3453134|BBa_K3453134]] page in the registry.
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===References===
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[1] Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell (2014) 159, 925-939.
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[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.
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[3] Salis HM. The ribosome binding site calculator. Methods in Enzymology (2011) 498: 19–42.
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[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.
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Latest revision as of 21:21, 26 October 2020


Rosewood DmRbcL Toehold Switch 2.1

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).


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

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. 

...............((((((((((..((((((...............))))))..))))))))))..((....))..(((((.....)))))...
T--Evry Paris-Saclay--RNAstructure p70 DmRbcL2-1.png

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_K3453044.

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_K3453134.

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_K3453134 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]