Difference between revisions of "Part:BBa K2206010"

 
 
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<partinfo>BBa_K2206010 short</partinfo>
 
<partinfo>BBa_K2206010 short</partinfo>
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Toehold switches are synthetic riboregulators that regulate gene expression post-transcriptionally. Gene expression can be activated in the presence of a cognate single stranded RNA molecule that contains an arbitrary sequence (the trigger RNA). The trigger RNA binds to the switch through base pairing, causing a conformational change that results in translation of the downstream protein coding region.  
 
Toehold switches are synthetic riboregulators that regulate gene expression post-transcriptionally. Gene expression can be activated in the presence of a cognate single stranded RNA molecule that contains an arbitrary sequence (the trigger RNA). The trigger RNA binds to the switch through base pairing, causing a conformational change that results in translation of the downstream protein coding region.  
  
The trigger RNA sequence can be separated into different RNA molecules. Hybridisation of these different RNA molecules results in a complete trigger RNA sequence, that is capable of activating the toehold switch.  
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The trigger sequence can be split into different RNA molecules. Colocalization of the split trigger parts results in a complete trigger RNA that is capable of activating the toehold switch.
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BBa_K2206011 and hsa-miR-27b-3p contain roughly half of the trigger sequence each. Colocalization of the split trigger RNA sequences results in a functional trigger RNA,  capable of activating the toehold switch. The formation of the duplex occurs through a highly specific binding step which results in single-base mismatch specificity. This part codes for a toehold switch that has a region complementary to the complete trigger RNA sequence formed of BBa_K2206011 and hsa-miR-27b-3p Therefore, this part can be used to regulate the expression of any protein in response to presence of both BBa_K2206011 and hsa-miR-27b-3p.
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For For more information, see the section on second series of toehold switches on the design page of CLSB-UK's 2017 wiki - http://2017.igem.org/Team:CLSB-UK/Design.
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We recommend using this part to regulate the expression of a reporter protein. This allows for quantification of hsa-miR-27b-3p. Our simulations show that this part would be better suited for hsa-miR-27b-3p quantification than our original toehold switch BBa_K2206000 due to its increased specificity and on state activity.
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We found that the transcripts produced by our other toehold switch parts were toxic to E.coli. We therefore recommend using this part with a non-leaky promoter. For reference, we found the part BBa_K808000 was too leaky when regulating the expression of one of our previous toehold switches.
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This part contains a strong RBS sequence.
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{{Template:CLSB-UK 17 Images 27b3p 2}}
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===Information contributed by City of London UK (2021)===
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[[File:ToeholdTools.png|x200px|center]]
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This toehold switch was characterized <i>in silico</i> using the ToeholdTools project that our team developed.
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See https://github.com/lkn849/thtools for more information.
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Metadata:
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*'''Group:''' City of London UK 2021
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*'''Author:''' Lucas Ng
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*'''Summary:''' Used our software ToeholdTools to investigate the target miRNA specificity and activation of this part.
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Raw data:
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*[[Media:BBa_K2206010_thtest.txt]]
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*[[Media:BBa_K2206010_crt.txt]]
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This contribution was autogenerated by the script '''contrib.py''', available at https://github.com/lkn849/thtools/tree/master/registry.
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----
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This switch was designed to detect the miRNA hsa-miR-27b-3p at a temperature of 37°C.
 +
We tested it against every mature <i>Homo sapiens</i> miRNA in miRBase and our analysis shows that at this temperature it is best used to detect hsa-miR-27b-3p.
  
Bba_X and hsa-miR-15b-5p contain roughly half of the trigger sequence each. Colocalization of the split trigger RNA sequences results in a functional trigger RNA that is capable of activating the toehold switch. This part codes for a toehold switch that contains a region that is complementary to the complete trigger RNA sequence formed of Bba_X and hsa-miR-15b-5p Therefore, this part can be used to regulate the expression of any protein in response to presence of both Bba_X and hsa-miR-15b-5p.
+
With hsa-miR-27b-3p at 37°C, the switch has a specificity of 1 ± 100 % and an activation of 13 ± 7 %.
 +
These values represent 95% confidence limits (z=1.96).
  
We found that the transcripts produced by our other toehold switch parts were toxic to E.coli. We therefore recommend using this part with a non-leaky promoter (for reference, we found K808000 to be too leaky).
+
The temperature&ndash;activation&ndash;specificity relationship is shown here.
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CRT is an acronym for CelsiusRangeTest, the class in our Python library responsible for the following graph:
  
N.B. This part contains a strong RBS sequence.
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[[File:BBa_K2206010_graph.png|500px|center]]
  
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Error bars represent the standard deviation.
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The line of best fit was calculated using a univariate cubic spline weighted inverse to each point's standard error.
  
  

Latest revision as of 10:26, 8 October 2021

Toehold switch for hsa-miR-27b-3p (second series)

Toehold switches are synthetic riboregulators that regulate gene expression post-transcriptionally. Gene expression can be activated in the presence of a cognate single stranded RNA molecule that contains an arbitrary sequence (the trigger RNA). The trigger RNA binds to the switch through base pairing, causing a conformational change that results in translation of the downstream protein coding region.

The trigger sequence can be split into different RNA molecules. Colocalization of the split trigger parts results in a complete trigger RNA that is capable of activating the toehold switch.

BBa_K2206011 and hsa-miR-27b-3p contain roughly half of the trigger sequence each. Colocalization of the split trigger RNA sequences results in a functional trigger RNA, capable of activating the toehold switch. The formation of the duplex occurs through a highly specific binding step which results in single-base mismatch specificity. This part codes for a toehold switch that has a region complementary to the complete trigger RNA sequence formed of BBa_K2206011 and hsa-miR-27b-3p Therefore, this part can be used to regulate the expression of any protein in response to presence of both BBa_K2206011 and hsa-miR-27b-3p.

For For more information, see the section on second series of toehold switches on the design page of CLSB-UK's 2017 wiki - http://2017.igem.org/Team:CLSB-UK/Design.

We recommend using this part to regulate the expression of a reporter protein. This allows for quantification of hsa-miR-27b-3p. Our simulations show that this part would be better suited for hsa-miR-27b-3p quantification than our original toehold switch BBa_K2206000 due to its increased specificity and on state activity.

We found that the transcripts produced by our other toehold switch parts were toxic to E.coli. We therefore recommend using this part with a non-leaky promoter. For reference, we found the part BBa_K808000 was too leaky when regulating the expression of one of our previous toehold switches.

This part contains a strong RBS sequence.

NUPACK Structure Analysis

RBSStart codonTrigger Binding SiteFree energy of secondary structure: -14.08 kcal/molACGUMFE structure at 35.0°Canti-miRNAmiRNAFree energy of secondary structure: -21.70 kcal/molACGUMFE structure at 35.0°C

anti-miRNAmiRNATriggersitebindingRBSStartcodonFree energy of secondary structure: -72.09 kcal/molACGUMFE structure at 35.0°C

Information contributed by City of London UK (2021)

ToeholdTools.png

This toehold switch was characterized in silico using the ToeholdTools project that our team developed. See https://github.com/lkn849/thtools for more information.

Metadata:

  • Group: City of London UK 2021
  • Author: Lucas Ng
  • Summary: Used our software ToeholdTools to investigate the target miRNA specificity and activation of this part.

Raw data:

This contribution was autogenerated by the script contrib.py, available at https://github.com/lkn849/thtools/tree/master/registry.


This switch was designed to detect the miRNA hsa-miR-27b-3p at a temperature of 37°C. We tested it against every mature Homo sapiens miRNA in miRBase and our analysis shows that at this temperature it is best used to detect hsa-miR-27b-3p.

With hsa-miR-27b-3p at 37°C, the switch has a specificity of 1 ± 100 % and an activation of 13 ± 7 %. These values represent 95% confidence limits (z=1.96).

The temperature–activation–specificity relationship is shown here. CRT is an acronym for CelsiusRangeTest, the class in our Python library responsible for the following graph:

BBa K2206010 graph.png

Error bars represent the standard deviation. The line of best fit was calculated using a univariate cubic spline weighted inverse to each point's standard error.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 65