Difference between revisions of "Part:BBa J119449"

 
 
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<partinfo>BBa_J119449 short</partinfo>
 
<partinfo>BBa_J119449 short</partinfo>
  
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The N Clone of Mammal Ribozyme is a riboswitch that cleaves itself in the presence of theophylline. It has a five nucleotide base change from the Mammal Ribozyme (J119446). It is cloned into the pSB1A2-BR vector, allowing for gene expression using a T7 promoter. The sequence for the original Mammal Ribozyme is found in Massively Parallel RNA Device Engineering in Mammalian Cells with RNA-Seq (https://www.nature.com/articles/s41467-019-12334-y).
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The N Clone of Mammal Ribozyme is a riboswitch that cleaves itself in the presence of theophylline. It has a five nucleotide (nt) base change from the Mammal Ribozyme (J119446). At nt 77-81, the original ribozyme sequence of CATAA was edited to AATAA.  It is cloned into the pSB1A2-BR vector, allowing for gene expression using a T7 promoter. The sequence for the original Mammal Ribozyme is found in Massively Parallel RNA Device Engineering in Mammalian Cells with RNA-Seq (https://www.nature.com/articles/s41467-019-12334-y).
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_J119449 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_J119449 SequenceAndFeatures</partinfo>
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<hr/>
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<h3>Charcterization and improvement in 2021 by SJTU-BioX-Shanghai</h3>
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In 2021, SJTU-BioX-Shanghai improved the sensibility of this part, see <html><a href="https://parts.igem.org/Part:BBa_K3714004">BBa_K3714004</a></html> for more details. In short, we characterized and improved the performance  (cleave fraction) with theophylline present <i>in vitro</i> by introducing some mutation in the loops of aptazyme[1]. <br/>
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[[File:K3714004-2.jpg]]
  
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<center><b>Figure 1| Improvement of BBa_J119449. a.</b>Cleavage properties of two aptazymes under a lower theophylline concentration gradient. Control means no theophylline was added. <b>b. </b>A diagram showing the intensity percentage of the cleaved band of each lane in <b>a</b>. Intensity was measured by ImageJ.</center>
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<b>Reference</b><br/>
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Townshend, B., Xiang, J. S., Manzanarez, G., Hayden, E. J. & Smolke, C. D. A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors. Nature Communications <b>12</b>, 1437, doi:10.1038/s41467-021-21716-0 (2021).
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_J119449 parameters</partinfo>
 
<partinfo>BBa_J119449 parameters</partinfo>
 
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Latest revision as of 00:58, 22 October 2021


N Clone of Mammal Ribozyme

The N Clone of Mammal Ribozyme is a riboswitch that cleaves itself in the presence of theophylline. It has a five nucleotide (nt) base change from the Mammal Ribozyme (J119446). At nt 77-81, the original ribozyme sequence of CATAA was edited to AATAA. It is cloned into the pSB1A2-BR vector, allowing for gene expression using a T7 promoter. The sequence for the original Mammal Ribozyme is found in Massively Parallel RNA Device Engineering in Mammalian Cells with RNA-Seq (https://www.nature.com/articles/s41467-019-12334-y).

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]

Charcterization and improvement in 2021 by SJTU-BioX-Shanghai

In 2021, SJTU-BioX-Shanghai improved the sensibility of this part, see BBa_K3714004 for more details. In short, we characterized and improved the performance (cleave fraction) with theophylline present in vitro by introducing some mutation in the loops of aptazyme[1].
K3714004-2.jpg

Figure 1| Improvement of BBa_J119449. a.Cleavage properties of two aptazymes under a lower theophylline concentration gradient. Control means no theophylline was added. b. A diagram showing the intensity percentage of the cleaved band of each lane in a. Intensity was measured by ImageJ.

Reference
Townshend, B., Xiang, J. S., Manzanarez, G., Hayden, E. J. & Smolke, C. D. A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors. Nature Communications 12, 1437, doi:10.1038/s41467-021-21716-0 (2021).