Team:Berlin diagnostX/Results
Results
Throughout our research project we developed Standard Operating Procedures, that are presented in the protocols section and an in silico sensor design algorithm that is described in the design section. These process innovations led to the subsequent optimization of a well performing sensor for Taenia Solium:
In the first series of screenings we reproduced published data of sensor 27B from the 2016 Pardee et al paper describing a toehold switch for Zika Virus:
We tested a first series of sensors that were designed by predicting the secondary structure of possible sensors and then selecting sensors with a low normalized ensemble defect. A low normalized ensemble defect indicates that a potential sensor spontaneously forms a secondary structure that is similar to the secondary structure of a toehold switch. This design principle led to sensors that did not show unspecific reaction, but that neither reacted upon adding trigger RNA. Trigger RNA was the only published RNA-Sequence of T. solium: TSO_31
In the next series of sensors tested, we replaced the selection based on the normalized ensemble defect with the score calculation mentioned in the design section. This led to a significant improvement of the sensor opening. At the same time, sensors showed a quite high degree of unspecific reactions.
Upon identifying a new, unique and highly expressed RNA molecule in the transcriptome of Taenia Solium, we created a new series of sensors aiming at this new target without changing the design algorithm. This approach led to two promising sensor candidates that were further analyzed:
Transcriptome
We obtained raw RNAseq data of Taenia Solium from GEO database (GSM2227058) and mapped it against a published genome of T. solium (PRJNA170813). In this way we identified 215 potential targets for sensor development. The interactive map on the left shows these targets. The darker the dot, the higher the expression level. Upon clicking on a dot, absolute expression level, Coding Sequence, information on splicing and GeneDB identifier is displayed. You can download the underlying data in an excel file here.
In our analysis we ranked the 215 potential RNA targets by their expression level. The target with the highest expression level was chosen for further sensor development: TsM_000297600.
Nested PCR
Fig. 1: Agarose gel of the first nested PCR attempt with the nested template and two toehold primers. PCR products 1 and 3 show the expected length, while lane 2 and 4 show, as anticipated, the failure of the negative controls. 1-nested PCR of switch 898 2-negative control of switch 898 (without P1) 3-nested PCR of switch 16 4-negative control of switch 16 (without P1)
