Team:Berlin diagnostX/Design
Design
The vision of diagnost-x
Developing a low cost, easy-to-handle diagnostic test for infection with the tapeworm Taenia solium
Transcriptome analysis of T.solium
Behind the Bioinformatics
Basics: RNA toehold switch
Toehold switches are RNA molecules that can regulate the downstream translation (in our case of a reporter protein) depending on the presence or absence of specific trigger RNA.
They consist of:
- a specific single-stranded toehold sequence
- a ribosome-binding site (RBS)
- a coding sequence for the reporter protein.
The reporter protein can only be produced if the toehold domain (recognition site) of the sensor has bound to its specific trigger RNA sequence. In absence of trigger RNA, the toehold switch forms a hairpin structure that prevents the ribosome from binding to the RBS, and thereby the translation (production of the reporter protein) can’t be initiated.
However, an RNA sensor alone is not sufficient for a screening test. Naked RNA cannot produce a protein nor induce a colour change – we thus relied on cell-free expression systems, a synthetic alternative to cell culture providing both easy handling as well as a controlled micro-environment for synthetic biology.
Basics: Cell free expression system
A cell free expression system is a system allowing for the production of protein using biological machinery without the use of living cells. The in vitro protein synthesis environment is highly controllable and more stable in the face of changes in temperature, buffers or similar interfering factors.
In order to establish our diagnostic test we thus needed:
- An RNA sensor designed to target the tapeworm
- A cell free expression system able to produce a color change
- T. solium specific trigger RNA
- (for field use: a carrier system to make the cell-free expression system storable)
We thus divided the work over three subteams:
Preliminary Data (Bioinformatics)
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Team 0: In-silico RNA sensor design
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Team 1: Sensor Synthesis
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Team 2: Sensor Screening/Cell free expression system
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Summary
In order to develop a diagnostic tool for the tapeworm infection Taeniasis, we developed a pipeline that allowed for designing, synthesizing and testing any given sensor within 2 days.
Step 1: In silico design
- Design of a toehold switch sensor specific to T. solium RNA
- Generating primers necessary for switch synthesis
Step 2: DNA-Switch Synthesis
- By using extension PCR or nested PCR, the toehold switches were synthesized
- Reporter gene (Beta-galactosidase) and T7 promoter were added to the sensor
Step 3: Sensor Screening
- Transcription of DNA sensor into RNA takes place in CFE system
- Specific binding of RNA sensor to trigger RNA leads to translation of reporter gene in CFE system
- By measuring absorption at 560 nm, color change was measured and enzymatic reaction quantified
Splitting the work into three teams enabled us to work continuously – trouble shooting in one team did not stall work in the other teams. Alongside the work on the core piece – a CFE system containing a sensitive and specific sensor – we tried to keep the ultimate goal in mind: a test usable in the field.
We thus investigated fixing the system onto cellulose membranes, making it storable, durable and easily transportable. Collaborations with partners in Berlin, Europe and India enabled us to further adapt our system to challenges of real-life application.
References
[1] Pardee, K. et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell 165, 1255–1266 (2016).
[2] Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell 159, 925–939 (2014)
[3] Gomez, S. et al. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR). Sci. Rep. 5, 9683 (2015)
