Difference between revisions of "Part:BBa K2206001"

Latest revision as of 10:21, 8 October 2021

Toehold switch for hsa-miR-27b-3p

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.

This part codes for a toehold switch that contains a region that is complementary to the microRNA hsa-miR-27b-3p (the trigger RNA). In the presence of hsa-miR-27b-3p, translation of the downstream protein coding region occurs.

This part can be used to regulate the expression of any protein in response to the levels of hsa-miR-27b-3p present in a system.

The transcripts produced by this part are toxic to E. coli. Therefore, we recommend the use of an inducible non-leaky promoter to allow for amplification in E. coli. For reference, we found Bba_K808000 was too leaky.

This part contains a strong RBS sequence.

NUPACK Structure Analysis

Information contributed by City of London UK (2021)

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-4999-5p.

With hsa-miR-4999-5p at 37°C, the switch has a specificity of 11 ± 200 % and an activation of 7 ± 5 %. 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:

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.

Caveats:

As per the above, we cannot confirm that this switch accurately detects the desired miRNA sequence.
The miRNA most targeted by this switch heavily fluctuates based on temperature.Therefore, we cannot confirm the reliability of this switch.

We do not recommend this part for future usage.

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]

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K2206001 short</partinfo>
-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, resulting in translation of the 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.
-This part codes for a toehold switch that contains a region that is complementary to the micro-RNA hsa-mir-27b-3p. In the presence of hsa-mir-27b-3p, translation of the downstream protein coding region occurs.
-This part can be used to regulate the expression of any protein in response to the presence of hsa-mir-27b-3p. This part contains a strong RBS. We believe that the transcripts produced by this part are toxic when expressed at reasonably high concentrations in E.coli (e.g. the concentrations produced from the BBa_J23111 constitutive promoter).
+This part codes for a toehold switch that contains a region that is complementary to the microRNA hsa-miR-27b-3p (the trigger RNA). In the presence of hsa-miR-27b-3p, translation of the downstream protein coding region occurs.
+This part can be used to regulate the expression of any protein in response to the levels of hsa-miR-27b-3p present in a system.
+The transcripts produced by this part are toxic to E. coli. Therefore, we recommend the use of an inducible non-leaky promoter to allow for amplification in E. coli. For reference, we found Bba_K808000 was too leaky.
+This part contains a strong RBS sequence.
+{{Template:CLSB-UK 17 Images 27b3p 1}}
+===Information contributed by City of London UK (2021)===
+[[File:ToeholdTools.png|x200px|center]]
+This toehold switch was characterized <i>in silico</i> 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:
+*[[Media:BBa_K2206001_thtest.txt]]
+*[[Media:BBa_K2206001_crt.txt]]
+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 <i>Homo sapiens</i> miRNA in miRBase and our analysis shows that at this temperature it is best used to detect hsa-miR-4999-5p.
+With hsa-miR-4999-5p at 37°C, the switch has a specificity of 11 ± 200 % and an activation of 7 ± 5 %.
+These values represent 95% confidence limits (z=1.96).
+The temperature&ndash;activation&ndash;specificity relationship is shown here.
+CRT is an acronym for CelsiusRangeTest, the class in our Python library responsible for the following graph:
+[[File:BBa_K2206001_graph.png|500px|center]]
+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.
+'''Caveats:'''
+*As per the above, we cannot confirm that this switch accurately detects the desired miRNA sequence.
+*The miRNA most targeted by this switch heavily fluctuates based on temperature.Therefore, we cannot confirm the reliability of this switch.
+We do not recommend this part for future usage.
 <!-- Add more about the biology of this part here