Summary of Results
In order to realise the project aptasense, we first developed, optimised and tested the building blocks of our project separately before assembling them all. The first part to develop was the cell-free technique in order to express our reporter gene. In the mean time, a part of the team learned how to make chips for microfluidics and use them for assays. Then we worked on testing the Zika toehold in lysates and studying the binding capacity and specificity of aptamers on thrombin and PDGF using microfluidics. At the same time we started developing our software Toehold Designer. To complement the microfluidics experiments, we started performing assays with beads in parallel. Finally, when all these segments of our project were studied and documented, we could finally work on the assembly of the segments. After making sure that the different blocks that make up our project worked separately, we went on to put them together and test whether protein can be detected and a signal generated in one setting.
In order to chronologically summarize our best accomplishments and discoveries during the elaboration of our project, here is a list:
- Successful production of functional lysates from different cell strains.
- Determination of which lysate is the best for protein production.
- Characterization of GFP-mut3b and EGFP in cell-free expression system.
- Successfully shown the positive effect for protein expression upon adding GamS.
- Successfully exhibited the two ways of adding gamS to the cell-free reaction: by adding purified GamS or by mixing the lysate with Top10-GamS.
- Determination of the best quantity of Top10-GamS to add to the lysate reaction.
- Discovery of the importance of T7 RNA polymerase and thus the discovery of a limiting factor for protein expression.
- Successfully tested lysates with lacZ reporter and determined which one was the best.
- Successful α-complementation.
- Successful lyophilized cell-free reactions.
- Thrombin Aptamers 1, 2 and 2 with extension bind α-Human Thrombin in buffer.
- Thrombin Aptamers 1 and 2 can detect α-Human Thrombin in a sandwich assay in buffer.
- Thrombin Aptamers 1 and 2 with extension can detect α-Human Thrombin in a sandwich assay in buffer.
- Thrombin can be detected in Human serum using both thrombin aptamers 1 and 2 with extension.
- PDGF Aptamers 1 and 2 can detect PDGF in a sandwich assay in buffer.
Magnetic beads accomplishments:
- Confirm that thrombin and PDGF can successfully be bound by 2 different aptamers simultaneously.
- Prove that fluorescence detection is a viable testing method.
- Prove that thrombin detection can be decreased to at least a 50 nM concentration.
- Prove that the entire aptamer 1-target protein-aptamer 2 with trigger complex can successfully trigger toeholds in lysate.
- Prove that proteins in their naturally occurring form can be isolated in cell free medium and subsequently detected.
With all this accomplished, we finally could test the assembly of all our project parts !
In the end, two experiments finalised our project: magnetic bead sandwich assays which combine aptamers and toeholds for protein detection. These experiments prove that magnetic bead sandwich assays inducing toehold activation by aptamer trigger extension in lysate are a viable option to detect naturally occurring proteins and support our initial hypothesis. Nevertheless, further testing is necessary to improve this process.