Difference between revisions of "Team:Berlin diagnostX/Results"
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Revision as of 02:17, 2 November 2017
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 first predicting the secondary structure of possible sensors and then selecting sensors with a low normalized ensemble defect. A low normalized ensemble defect indicated 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 leak, but that neither reacted upon adding trigger RNA:
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 leakiness.
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:
Analysis with more replicates confirmed the good performance of our two sensor candidates. Colour change was not only visible in measurements using the GloMax, but also with the naked eye
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)
