Difference between revisions of "Part:BBa K3806008"
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===Usage and Biology===
 
===Usage and Biology===
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Ligand-dependent self-cleaving ribozymes, also known as aptazymes, have emerged in recent years as valuable tools to control gene expression [1]. Aptazymes can be programmed to respond to a wide range of small-molecule ligands with high sensitivity and selectivity. They therefore have a large number of potential applications, ranging from disease diagnosis, prognosis, or treatment to detecting small pollutants in the environment. Synthetic genetic switches are a promising tool to detect and quantify small molecules. Ligand regulated self-cleaving ribozymes are of special interest as cleavage of the aptazyme can be coupled to regulated gene expression in vivo or in vitro. Moreover, newly developed methods such as DRIVER (de novo rapid in vitro evolution of RNA biosensors) enable rapid, automated, and multiplexed engineering of aptazymes sequences to diverse ligands[2].
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Ligand-dependent self-cleaving ribozymes, also known as aptazymes, have emerged in recent years as valuable tools for controlling gene expression [2]. Therefore, aptazymes present a large number of potential applications, ranging from disease diagnosis, prognosis, or treatment to detecting small pollutants in the environment. Aptazymes can be programmed to respond to a wide range of small-molecule ligands with high sensitivity and selectivity. Newly developed methods such as DRIVER (de novo rapid in vitro evolution of RNA biosensors) enable rapid, automated, and multiplexed engineering of aptazymes sequences to diverse small molecules [1].  
  
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Aptazymes are a class of engineered riboswitches that control gene expression by small molecule-induced self-cleavage of a ribozyme
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In the study of Townshed et al. [1], DRIVER was used to evolve a theophylline binding aptazyme that undergoes self-cleavage in the absence of theophylline and remains uncleaved in its presence (Fig. 1). The TU Delft iGEM team 2021: <i>(i)</i> characterized the cleavage of this aptazyme using a Urea-PAGE gel following a co-transcriptional cleavage assay. <i>(ii)</i> showed the value of this part when used as a positive control, and <i>(iii)</i> proved that this part (<HTML><a href="https://parts.igem.org/Part:BBa_K3806018" target="_blank"><b>BBa_K3806018</b></a></HTML>) can be used to regular gene expression  <i>in vitro</i> (<HTML><a href="https://parts.igem.org/Part:BBa_K3806014" target="_blank"><b>BBa_K3806014</b></a></HTML> and <HTML><a href="https://parts.igem.org/Part:BBa_K3806016" target="_blank"><b>BBa_K3806016</b></a></HTML>).
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The study of Townshed et al. [2] created a theophylline binding aptazyme that is evolved to undergo self-cleavage in the absence of theophylline and preserves its structure in the presence of theophylline. The TU Delft 2021 team characterized the cleavage of this aptazyme on an Urea-PAGE gel. This part is extensively used in the engineering of vitamin binding aptazymes as control for each DRIVER round.
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The TU Delft 2021 team provides the iGEM community with a novel genetic switch construct (BBa_K3806016) in which an aptazyme sequence is fused to the <i>lacZ</i> reporter gene for converting ligand concentration to a colorimetric read-out. The BBa_K3806016 contains a theophylline-binding aptazyme sequence [1], yet this part is designed to be modular and serve as a template to engineer other ligand-specific genetic circuits.
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[[File:T--TUDelft--Aptazyme.png|500px|center|]]
 
[[File:T--TUDelft--Aptazyme.png|500px|center|]]
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<HTML><b>Fig. 1 Theophylline-binding aptazyme (A) 2D structure, and (B) predicted 3D structure.</b> The structure of the aptazyme resembles that of the sTRSV hammerhead ribozyme. It is expected that the binding of the ligand to specific sequences within the large loop (30 nucleotides), affects the interactions with the smaller loop (7 nucleotides), thereby hindering self-cleavage  The cleavage site is indicated with a red arrow.</html>
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<HTML><b>Fig. 1 Theophylline-binding aptazyme (A) 2D structure, and (B) predicted 3D structure.</b> The structure of the aptazyme resembles that of the sTRSV hammerhead ribozyme. It is expected that the binding of the ligand to specific sequences within the large loop (30 nucleotides), affects the interactions with the smaller loop (7 nucleotides), hindering self-cleavage  The cleavage site is indicated with a red arrow.</html>
  
  

Revision as of 11:22, 21 October 2021


Theophylline-binding aptazyme

Positive control for DRIVER

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]

Usage and Biology

Ligand-dependent self-cleaving ribozymes, also known as aptazymes, have emerged in recent years as valuable tools for controlling gene expression [2]. Therefore, aptazymes present a large number of potential applications, ranging from disease diagnosis, prognosis, or treatment to detecting small pollutants in the environment. Aptazymes can be programmed to respond to a wide range of small-molecule ligands with high sensitivity and selectivity. Newly developed methods such as DRIVER (de novo rapid in vitro evolution of RNA biosensors) enable rapid, automated, and multiplexed engineering of aptazymes sequences to diverse small molecules [1].

In the study of Townshed et al. [1], DRIVER was used to evolve a theophylline binding aptazyme that undergoes self-cleavage in the absence of theophylline and remains uncleaved in its presence (Fig. 1). The TU Delft iGEM team 2021: (i) characterized the cleavage of this aptazyme using a Urea-PAGE gel following a co-transcriptional cleavage assay. (ii) showed the value of this part when used as a positive control, and (iii) proved that this part (BBa_K3806018) can be used to regular gene expression in vitro (BBa_K3806014 and BBa_K3806016).


T--TUDelft--Aptazyme.png

Fig. 1 Theophylline-binding aptazyme (A) 2D structure, and (B) predicted 3D structure. The structure of the aptazyme resembles that of the sTRSV hammerhead ribozyme. It is expected that the binding of the ligand to specific sequences within the large loop (30 nucleotides), affects the interactions with the smaller loop (7 nucleotides), hindering self-cleavage The cleavage site is indicated with a red arrow.


References

  1. [1] Zhong, G., Wang, H., Bailey, C. C., Gao, G., & Farzan, M. (2016). Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells. eLife, 5, e18858.
  2. [2] Townshend, B., Xiang, J. S., Manzanarez, G., Hayden, E. J. and Smolke, C. (2021). A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors. Nat Commun, 12, 1437.