Difference between revisions of "Part:BBa K4195181"
 
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This sequence is the guide.<br/>
 
This sequence is the guide.<br/>
 
===Ribozyme ENabled Detection of RNA (RENDR)===
 
===Ribozyme ENabled Detection of RNA (RENDR)===
−
RENDR is a high-performing, plug-and-play RNA-sensing platform[1]. RENDR utilizes a split variant of the ''Tetrahymena thermophila'' ribozyme by synthetically splitting it into two non-functional fragments (Fig. 1). Two fragments are each appended with designed RNA guide sequences, which can interact with the RNA input of interest. The split ribozyme is then inserted within a desired gene output. When bound with the RNA input, two transcribed split ribozyme fragments are triggered to self-splice and thus the intact transcript of the protein output will form.<br/>
+
RENDR is a high-performing, plug-and-play RNA-sensing platform(<i>1</i>). RENDR utilizes a split variant of the ''Tetrahymena thermophila'' ribozyme by synthetically splitting it into two non-functional fragments (Fig. 1). Two fragments are each appended with designed RNA guide sequences, which can interact with the RNA input of interest. The split ribozyme is then inserted within a desired gene output. When bound with the RNA input, two transcribed split ribozyme fragments are triggered to self-splice and thus the intact transcript of the protein output will form.<br/>
 
[[File:T--XMU-China--comp.png|400px]]<br/>
 
[[File:T--XMU-China--comp.png|400px]]<br/>
 
'''Fig. 1 Schematic illustration of RENDR.'''<br/>
 
'''Fig. 1 Schematic illustration of RENDR.'''<br/>
 +
 
===Usage and Design===
 
===Usage and Design===
 
We replicate the circuit used in the literature as reference, and separately designed two split ribozymes as different parts <partinfo>BBa_K4195073</partinfo> and <partinfo>BBa_K4195072</partinfo>. The combined one (<partinfo>BBa_K4195181</partinfo>) was assembled into the vector pSB1C3 by standard BioBrick assembly. After transcription, two RNA guides can interact with each other. <br/>
 
We replicate the circuit used in the literature as reference, and separately designed two split ribozymes as different parts <partinfo>BBa_K4195073</partinfo> and <partinfo>BBa_K4195072</partinfo>. The combined one (<partinfo>BBa_K4195181</partinfo>) was assembled into the vector pSB1C3 by standard BioBrick assembly. After transcription, two RNA guides can interact with each other. <br/>
 
===Characterization===
 
===Characterization===
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Agarose Gel Electrophoresis<br/>
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=====Agarose Gel Electrophoresis=====
 
<partinfo>BBa_K4195181</partinfo> was assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmids were transformed into ''E. coli'' BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.<br/>
 
<partinfo>BBa_K4195181</partinfo> was assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmids were transformed into ''E. coli'' BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.<br/>
 
[[File:T--XMU-China--comp g1α.png|400px]]<br/>
 
[[File:T--XMU-China--comp g1α.png|400px]]<br/>
−
Fig. 2 The result of colony PCR. Plasmid pSB1C3<br/>
+
'''Fig. 2 The result of colony PCR. Plasmid pSB1C3.'''<br/>
 +
 
 
===Reference===
 
===Reference===
−
[1]L. Gambill ''et al.'', https://www.biorxiv.org/content/10.1101/2022.01.12.476080v1 (2022).
+
1. L. Gambill ''et al.'' https://www.biorxiv.org/content/10.1101/2022.01.12.476080v1 (2022).
 +
 
 +
 
 +
 
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K4195181 SequenceAndFeatures</partinfo>

Latest revision as of 05:27, 13 October 2022

Biology

This sequence is the guide.

Ribozyme ENabled Detection of RNA (RENDR)

RENDR is a high-performing, plug-and-play RNA-sensing platform(1). RENDR utilizes a split variant of the Tetrahymena thermophila ribozyme by synthetically splitting it into two non-functional fragments (Fig. 1). Two fragments are each appended with designed RNA guide sequences, which can interact with the RNA input of interest. The split ribozyme is then inserted within a desired gene output. When bound with the RNA input, two transcribed split ribozyme fragments are triggered to self-splice and thus the intact transcript of the protein output will form.
T--XMU-China--comp.png
Fig. 1 Schematic illustration of RENDR.

Usage and Design

We replicate the circuit used in the literature as reference, and separately designed two split ribozymes as different parts BBa_K4195073 and BBa_K4195072. The combined one (BBa_K4195181) was assembled into the vector pSB1C3 by standard BioBrick assembly. After transcription, two RNA guides can interact with each other.

Characterization

Agarose Gel Electrophoresis

BBa_K4195181 was assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.
T--XMU-China--comp g1α.png
Fig. 2 The result of colony PCR. Plasmid pSB1C3.

Reference

1. L. Gambill et al. https://www.biorxiv.org/content/10.1101/2022.01.12.476080v1 (2022).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 233
    Illegal NheI site found at 828
    Illegal NheI site found at 1636
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 730
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 315
    Illegal BsaI.rc site found at 1555