Part:BBa_K3714004

Improved theophylline biosensor

An improved aptazyme responsing to theophylline, which means its self-cleaving activity can be inhibited by adding theophylline. This aptazyme shows higher sensibility than an existing part (BBa_J119449).

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]

---

By introducing mutations in loop Ⅰ and loop Ⅱ[1], we achieved an aptazyme with higher responsing fold and less leakage under high theophylline concentration (Fig.1). We characterized the responding fold of these aptazymes under diffrent theophylline concentrations by running the transcipt on a denatured PAGE. Cleave fraction was defined as the intensity of the Cleaved band / the sum of the Cleaved and Uncleaved band. While both aptazymes exhibit similar cleave fraction without theophylline(i.e. 6.2% for BBa_K3714004 and 5.8% for BBa_J119449),our improved aptazyme shows a higher cleave fraction with saturated theophylline (55%, while BBa_J119449 has 41%).

Figure 1 | Improvement of an existing theophylline aptazyme. Cleavage properties of two aptazymes under different theophylline concentration. The aptazymes were transcribed in vitro with certain amount of theophylline, and the products were run a a denatured PAGE. The number above each lane means dilution fold of saturated theophylline aqueous solution at 25℃.

Considering the further quantification of our engineering, and the working concentration where theophylline biosensors reside, we used a lower concentration gradient. Our aptazyme exhibits a 2-fold change of cleavage proportion at ~1mg/ml (0.125x dilution of saturated solution at room temperature) while the origin apatazyme (BBa_J119449) shows only 1.3-fold change (Fig.2a). We made a dose-response curve where our engineering outcome could be evaluated easier.

Figure 2 | Improvement of an existing theophylline aptazyme. 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).