Team:Michigan/Results

Results

Switch 1.0 was induced with minimal background with its corresponding DNA trigger. However, when induced with thrombin, with aptamer and trigger preincubated, GFP expression became sporadic and had a loose negative correlation with increasing thrombin concentration. Additionally, the negative controls (switch, trigger, aptamer, and no protein) showed the same induction as the positive controls. Switch 2.0 adapted to thrombin protein showed increasing GFP expression with increasing thrombin and minimal background as desired. However, Switch 2.0 adapted to gliadin had a negative control (switch and no protein) with GFP expression at the same rate as when induced with gliadin. Only Switches 1.0 and 2.0 had been tested at the point of writing. All switches are RNA, transcribed from constitutively expressed DNA, and all reporters are GFP, unless otherwise noted. All in vitro transcription and translation reactions were performed using an E. coli based, cell free expression kit (PURExpress In Vitro Protein Synthesis, New England BioLabs).

Figure 1: Toehold Switches with and without triggers
Switches G (forward engineered) and H (first generation) were adapted for thrombin detection with a GFP reporter was successfully induced by the corresponding DNA trigger, showing over 100,000 RFU, while the uninduced switch showed minimal background. Control switch 16 with GFP or Mcherry with approximately the same toehold design as G and H were tested. Switch 16 with GFP performed similarly, while Mcherry didn’t work well as a reporter. Switch G was adapted for the Switch 1.0 design.

Figure 2: Thrombin Titration (switch + trigger + aptamer + thrombin), 9/7/15 results
As thrombin concentration increases, GFP expression generally declines. The negative controls (switch + trigger + aptamer) show higher GFP expression than the positive control (switch + trigger). This experiment was repeated multiple times with the same result, see lab notebook.

Figure 3: Positive Control (switch + trigger) with thrombin titration, 9/7/15 results
If Switch 1.0 worked the way it was intended to, thrombin would have no impact on just switch and trigger in solution. After observing that the switch “induced” with 25uM thrombin had the lowest GFP expression, the effect of just thrombin on the Switch 1.0 adapted for thrombin detection was tested. The switch was induced at a constant concentration of trigger, with varying thrombin concentration. Switch 1.0 showed decreasing GFP expression loosely correlated with increasing concentrations of thrombin protein. This suggests that the thrombin may be able to bind to the RNA version of the aptamer that is completely exposed in the toehold region of the switch and Switch 2.0 was designed to take advantage of the this.

Figure 4: Switch 2.0 Induced with thrombin, 9/17/15 results
Switch 2.0 LINK TO DESIGN adapted for thrombin detection had increasing GFP expression with increasing concentrations of thrombin. This switch was designed so thrombin directly binds the exposed aptamer/toehold region, causing the hairpin to unfold so the ribosome binding site is exposed and translation can occur. Switches induced with thrombin had significantly higher expression than uninduced switches (0uM thrombin). While the highest concentration of thrombin failed to induce the switch, this is likely due to a failed reaction as this was the only reaction well used for this experiment that did not have condensation when the plate was removed from the reader (A8). Thus, Switch 2.0 GFP expression increased with increasing amounts of thrombin.

Figure 5: Switch 2.0 Induced with gliadin, 9/18/15 results
Switch 2.0 adapted to gliadin showed high GFP expression, regardless of gliadin concentration.

Discussion
Switch 1.0 was successfully induced with just trigger, but protein appeared to inhibit GFP expression. Additionally, while just Switch 1.0 in solution produced minimal background, trigger and aptamer together in solution with Switch 1.0 resulted in maximum measurable GFP expression. This implies that though the in vitro translation reactions are occurring correctly, the aptamer and trigger are unbound under the experimental conditions (no added salt, 37 degrees Celsius), allowing high background. Additionally, high concentrations of thrombin inhibit GFP expression, implying that thrombin is inhibiting translation or the plate reader reading. One possibility is that thrombin prevents the trigger from binding, by binding to the toehold region, which is the RNA version of the DNA aptamer.

Switch 2.0, adapted to the respective proteins, was successfully induced by thrombin protein, but not gliadin protein. Switch 2.0 showed an increase in GFP expression as thrombin protein concentration increased. It also responded at the lowest concentration tested, 0.2uM. The same switch adapted to the much longer gliadin protein showed consistent GFP expression, even without the presence of gliadin. The thrombin version Switch 2.0 shows much potential, and it seems promising that gliadin could show similar results if with optimization. The hairpin strength could be increased to prevent uninduced expression.

All together, the results of the Aptapaper project are promising. High GFP expression was seen with no optimization of experimental conditions, demonstrating the robustness of this system. While many different switch designs were discussed during planning, the results obtained generally only show one iteration of each switch. Binding strength of different parts of each switch could be adjusted to make them work over a wide range of proteins, optimizing the switch design.