Part:BBa_K3714000

Gardiquimod biosensor 337

The template of a new self-cleaving aptazyme[1], which keeps itself intact with gardiquimod present, and catalyzes the self-cleavage reaction in the absence of gardiquimod. This part could be used as a switch for gene expression controlled by gardiquimod. The feature "Aptazyme" is necessary, and two A-rich oligonucleotides are added at both terminal to avoid unwanted secondary structure.

Usage and Biology

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]

This aptazyme was found in a random oligonucleotide library designed based on ribozyme sTRSV. The two loops of native sTRSV were replaced by two random sequences, which might form an aptamer domain, affecting the activity of ribozyme. We intended to use this aptazyme to validate our design of an colloidal gold based detection platform. So we tested its activity in advance(Fig.1).

Figure.1 | Verification of biosensor gard-337. An in vitro transcription system was employed to test if this biosensor can response gardiquimod signal. The product was run on a denatured PAGE. Gardiquimod were added during transcription at 300μM for +target lane.

The cleave fraction of each lane was determined by ImageJ. The aptazyme showed a percentage of 77% cleaved without gardiquimod and 14% cleaved with 300μM gardiquimod. This performance is slightly different from the efficiency mentioned in literature[1], we contributed this difference to technical bias during measuring cleave fraction with PAGE. After validation, we engineered the A-rich sequences to two probes named BBa_K3714002.
Reference
[1] 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).