Team:EPFL
Project overview
Have a more in-depth look at our project and dicover the world of cell-free biology.
Educational Cell-Free Mini Kit
We created a cell-free mini kit in order to introduce the basic concepts of synthetic biology to high school students.
Software
We created an open-source software to design and evaluate new toehold sensors.
Actions that required a few days to complete now take only a few minutes!
With all this accomplished, we finally could test the assembly of all our parts !
We managed to do two experiments in order to prove the whole project: we wanted to trigger of the toehold with an aptamer using a magnetic bead sandwich assay
We performed magnetic bead pulldown assays in order to determine whether or not such an assay had the capacity to trigger the Zika toehold 27B in sufficient amounts to see a difference. After a pulldown assay, where biotinylated PDGF aptamer 1, PDGF and PDGF aptamer 2 extended with a trigger sequence were added consecutively. We resuspended the beads in lysate containing the DNA encoding the Zika toehold 27B uphold of the LacZ gene. After 5 hours of incubation, the beads were then separated from the lysate, which was deposited into a micro-well plate for subsequent absorbance measurements.
From the data extracted, we can see that the absorbance at 595 nm (optimal absorbance to measure the substrate turnover of β-Galactosidase) increases more or less correctly with the different types of assays performed. As we can see the sandwich assay does indeed express a higher absorbance of β-Galactosidase than controls without PDGF and with or without trigger. Yet, in the first experiment, we can see a problem as the control without PDGF and trigger expresses a higher absorbance than that with trigger (Fig. 1a). However, this discrepancy disappeared when we repeated the assay (Fig. 1b) with the toehold upstream of the LacZ gene rather than using the toehold upstream of the LacZα gene, which was used in the first experiment. Hence, we can say, with the available data, that magnetic bead sandwich assays inducing toehold triggering in lysate are a viable option to detect naturally occurring proteins, proving that our initial hypothesis was correct, even though further testing is necessary to further characterize this process.
The absorbance of β-Gal was measured using a plate-reader at a 595 nm wavelength (maximum absorbance of β-Gal). This histogram shows the difference in absorbance between the positive and negative controls (+ control, - control, resp.), the sandwich assay and its respective controls. The positive and negative controls contain lysate with toehold, with or without trigger respectively, but said trigger was directly added to the solution and not the result of a pulldown assay. On the other hand, the sandwich assay controls are labeled in accordance to the addition or not of trigger and PDGF. Hence, the sandwich assay control without PDGF and trigger is labeled No trigger, no PDGF, whereas that without PDGF but where trigger was added is labeled Trigger, no PDGF. Finally, the sandwich assay containing all components for a successful detection of PDGF is labeled PDGF + trigger.
The graph shows the difference in absorbance between the complete sandwich assay (PDGF + trigger), the control where PDGF was not added (Trigger, no PDGF) and the control where neither aptamer 2 extended with trigger nor PDGF were added (No trigger, no PDGF).
