Difference between revisions of "Part:BBa K3714004"
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K3714004 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3714004 SequenceAndFeatures</partinfo> | ||
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− | 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%). | + | 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%).</p> |
<center>[[File:K3714004-1.jpg]]</center> | <center>[[File:K3714004-1.jpg]]</center> | ||
Revision as of 00:25, 22 October 2021
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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%).
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.
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).