Part:BBa_J119449

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]

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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].

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).

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