T7-RBS-lacZ_Synthetic Toehold Switch TSM_62
Usage and Biology
T. solium is a tapeworm that is endemic in most parts of the world. An infection can cause serious symptoms up to epilepsy and blindness. There is currently no cheap point-of-care test available, impeding diagnosis of T. solium infections in affected areas. We use toehold sensors as an approach for a new test of T. solium infections.
The target for this toehold sensor is the reverse complement of the TsM_000297600 mRNA. Our analysis showed that this transcript is quite unique to T. solium and also highly expressed in the tapeworm. We modeled our toehold sensors to the reverse complement in order to detect NASBA amplified RNA. In presence of the target sequence, the sensor catalyses a color change from yellow to violet, allowing for macroscopic readout without additional devices.
Toehold switches are RNA molecules that can regulate the downstream translation (in our case of the reporter protein LacZ) depending on the presence or absence of specific trigger RNA. They consist of a specific single-stranded toehold sequence, a ribosome-binding site (RBS) and a sequence for the reporter protein.
The reporter protein can only be produced if the toehold domain (recognition site) of the sensor has bonded with its specific target RNA sequence. In absence of trigger RNA, the toehold switch forms a hairpin structure that prevents the ribosome from binding to the RBS, thereby the translation (production of a Protein from RNA) can’t be initiated.
The LacZ reporter protein is a beta-galactosidase. Once expressed it catalyses the hydrolysation of the β-glycosidic bond in our indicator molecule Chlorophenol Red-β-D-galactopyranoside inducing a color change from yellow to violet.
The DNA encoding for the toehold sensor is ligated to a T7-Promotor which allows expression in different E. coli strains but also in a cell free expression system. We characterised the switch and plan its deployment in a cell free expression system (PURExpress, NEB).
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
Unless otherwise specified all characterisation experiments were conducted using the same experimental design. The reaction volumes of 4µl were pipetted into 384 well plates (Greiner Bio One, #784101) which are suitable for measurement of low volumes. The plates were sealed with a RNAse-free translucent membrane and covered with a lid to prevent evaporation.
The total reaction of 4µl consisted of:
- 3µl cell free expression system (PURExpress, NEB) with RNA or water
- the composition of the cell free expression system followed the proportions recommended by the manufacturer (51% Solution A, 39% Solution B, 5% RNAsin, 5% Dye (Chlorophenol Red-β-D-galactopyranoside at 6.6mg/ml)
- either RNA (concentrated for a final concentration of 3000nM in the total volume of 4µl) or an equal volume of nuclease free water was added to the cell free expression system.
- as control RNA we used the Zika RNA specific to our TSO47 toehold sensor
- 1µl DNA with a concentration of 12nM which makes for a final concentration of 3nM DNA per 4µl reaction
The reaction output was measured in the Glomax Discovery (Promega) at 37°C. The absorption was measured at 560nm which is the value closest to the absorption maximum of Chlorophenol Red. Measurement was performed every five minutes for either two or four hours.