Difference between revisions of "Team:Michigan/Results"

 
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<h2>Results</h2>
 
<h2>Results</h2>
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<strong>Overview</strong><br>Two different switch designs, Switch 1.0 and Switch 2.0, were adapted for thrombin tested in vitro.  The faulty Switch 1.0 design inspired the Switch 2.0 design, which responded to thrombin at the lowest concentrations tested.  Thrombin was selected as a model protein because it has well characterized aptamers and is commercially available.  Switch activation was measured by GFP fluorescence, as GFP tends to have higher expression than other reporters when used with in vitro translation kits<sup>1</sup>. New England Biolabs PureEXPRESS kits and 50 ng of switch DNA were used for in vitro transcription and translation according to standard protocol, with the only additions being thrombin, aptamer, and trigger as dictated by the experiment.  To verify Switch 1.0 functionality, the switch and its corresponding DNA trigger were combined with in vitro translation kits, and GFP fluorescence was measured.  Switch 1.0 alone had low fluorescence, while Switch 1.0 with trigger had high fluorescence (Figure 1).  Switch 1.0 was then tested with thrombin induction, at varying concentrations of thrombin.  Trigger and aptamer were pre-incubated to allow them to hybridize and prevent unbound trigger from binding the toehold region and then added to the in vitro translation kits, switch DNA, and thrombin.  High expression was seen for all combinations, including switch with trigger and aptamer with 0 uM thrombin, with the one exception being 25 uM thrombin (Figure 2).  As this was the opposite of the desired result, Switch 1.0 was then tested without any aptamer.  This experiment showed that high concentrations of thrombin could be inhibiting the switch (Figure 3).  This inspired the Switch 2.0 design, where the protein binds directly to the switch in a different way, causing the hairpin to unfold and the switch to be turned on.  Switch 2.0 was tested with thrombin induction at varying concentrations of thrombin and fluorescence increased with increasing thrombin (Figure 4). 
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<p><br>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).</p>
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<div style="width: 700; height: 350; text-align: center;"><img src="https://static.igem.org/mediawiki/2015/3/36/SwitchThrombin.png" style="vertical-align: middle; width: 700; height: 350"></div>
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<div style="width: 700; height: 350"><img src="https://static.igem.org/mediawiki/2015/thumb/3/36/SwitchThrombin.png/800px-SwitchThrombin.png" style="vertical-align: middle; width: 700; height: 350"></div>
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<div id="small">
 
<div id="small">
 
<p><strong>Figure 1: Toehold Switches with and without triggers</strong><br>
 
<p><strong>Figure 1: Toehold Switches with and without triggers</strong><br>
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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.</div><br></p>
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Switch 1.0 was adapted for thrombin detection with a GFP reporter and induced by the corresponding DNA trigger, showing over 100,000 RFU, while the uninduced switch showed minimal background.
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</div><br></p>
  
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<div class="pic2"><img src="https://2015.igem.org/File:MichiganResults_2.png"></div>
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<div style="width: 700; height: 350; text-align: center;"><img src="https://static.igem.org/mediawiki/2015/thumb/e/e7/MichiganResults_2.png/800px-MichiganResults_2.png" style="vertical-align: middle; width: 700; height: 350"></div>
  
 
<div id="small">
 
<div id="small">
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<p><strong>Figure 2: Thrombin Titration (switch + trigger + aptamer + thrombin), 9/7/15 results</strong><br>
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<p><strong>Figure 2: Thrombin Titration (switch 1.0 + trigger + aptamer + thrombin), 9/7/15 results</strong><br>
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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.</div><br></p>
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The positive control, consisting of Switch 1.0  with trigger had high expression.  The negative control, consisting of Switch 1.0 with pre-incubated trigger and aptamer had high expression. Switch 1.0 with trigger, aptamer, and 0.2 uM-5.0 uM thrombin had high expression, and 25.0 uM had low expression, the opposite of the intended design.  This experiment was repeated several times with the same result.
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</div><br></p>
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<div style="width: 700; height: 350; text-align: center;"><img src="https://static.igem.org/mediawiki/2015/thumb/a/a6/MichiganResults_3.png/800px-MichiganResults_3.png" style="vertical-align: middle; width: 700; height: 350"></div>
  
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<div class="pic3"><img src="https://2015.igem.org/File:MichiganResults_3.png"></div>
 
  
 
<div id="small">
 
<div id="small">
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<p><strong>Figure 3: Positive Control (switch + trigger) with thrombin titration, 9/7/15 results</strong><br>
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<p><strong>Figure 3: Positive Control (switch + trigger, no aptamer) with thrombin titration, 9/7/15 results</strong><br>
 
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.</div><br></p>
 
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.</div><br></p>
  
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<div class="pic4"><img src="2015.igem.org/File:MichiganResults_4"></div>
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<div style="width: 700; height: 350; text-align: center;"><img src="https://static.igem.org/mediawiki/2015/thumb/9/9b/Results1.png/800px-Results1.png" style="vertical-align: middle; width: 700; height: 350"></div>
  
 
<div id="small">
 
<div id="small">
 
<p><strong>Figure 4: Switch 2.0 Induced with thrombin, 9/17/15 results</strong><br>
 
<p><strong>Figure 4: Switch 2.0 Induced with thrombin, 9/17/15 results</strong><br>
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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.</div><br></p>
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Switch 2.0 adapted for thrombin detection and induced with 0.2-5uM thrombin had increasing expression with increasing thrombin concentrations.  Negative control, consisting of Switch 2.0 with no thrombin, had minimal expression.  Blank, consisting of water, had minimal fluorescence.  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.</div><br></p>
  
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<div class="pic41"><img src="https://2015.igem.org/File:Pic4_1.jpeg"></div>
 
  
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<div class="pic5"><img src="https://2015.igem.org/File:Gliadin_titration.png"></div>
 
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<p><strong>Figure 5: Switch 2.0 Induced with gliadin, 9/18/15 results</strong><br>
 
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Switch 2.0 adapted to gliadin showed high GFP expression, regardless of gliadin concentration.</div><br></p>
 
  
 
<p>
 
<p>
 
<strong>Discussion</strong><br>
 
<strong>Discussion</strong><br>
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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.<br><br>
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Two different switch designs were tested with thrombin and Switch 2.0 responded successfully, even at the lowest concentration of thrombin tested.<br><br>
  
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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.<br><br>
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Switch 1.0 was successfully induced by the DNA trigger; however, 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 the aptamer and trigger are unbound under the experimental conditions (no added salt, 37 °C), allowing high background. Additionally, fluorescence is low at high concentrations of thrombin, implying that thrombin is inhibiting translation of GFP. One possibility is that thrombin inhibits switch activation by binding to the toehold region, which is the RNA version of the DNA aptamer.  This inspired the successful Switch 2.0 design.<br><br>
  
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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.<br><br>
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Switch 2.0 was successfully induced by thrombin. Switch 2.0 showed an increase in GFP expression as thrombin protein concentration increased and responded to the lowest concentration tested, 0.2uM. The thrombin version of Switch 2.0 shows much potential, and it seems promising that adapting the switch for other proteins could yield similar results.<br><br>
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</p>
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Altogether, 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 only show one iteration of each switch.  Thus, Switch 2.0 was successful with zero optimization of the design, and it is likely that performance could be further improved.  Binding strength of different parts of each switch (toehold to protein, hairpin) could be optimized to adapt Switch 2.0 for a wide range of proteins, while maintaining high reporter expression and low background.  <br><br>
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<strong>Future Directions</strong><br>
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- Adapt, test, and determine limit of detection of Switch 2.0 for a variety of proteins<br>
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- Optimize to increase reporter expression for Switch 2.0 and reduce background<br>
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- Test specificity using biological sample<br>
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- Demonstrate on paper using a colorimetric reporter<br>
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<br><br>
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<p>
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<p><strong>References</strong></p>
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<ol>
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<li>Pardee, K., Green, A., Ferrante, T., Cameron, D., Daleykeyser, A., Yin, P., & Collins, J. (n.d.). Paper-Based Synthetic Gene Networks. Cell, 940-954.
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</li>
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<br>
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</ol>
 
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</html>

Latest revision as of 00:21, 21 November 2015

Results

Overview
Two different switch designs, Switch 1.0 and Switch 2.0, were adapted for thrombin tested in vitro. The faulty Switch 1.0 design inspired the Switch 2.0 design, which responded to thrombin at the lowest concentrations tested. Thrombin was selected as a model protein because it has well characterized aptamers and is commercially available. Switch activation was measured by GFP fluorescence, as GFP tends to have higher expression than other reporters when used with in vitro translation kits1. New England Biolabs PureEXPRESS kits and 50 ng of switch DNA were used for in vitro transcription and translation according to standard protocol, with the only additions being thrombin, aptamer, and trigger as dictated by the experiment. To verify Switch 1.0 functionality, the switch and its corresponding DNA trigger were combined with in vitro translation kits, and GFP fluorescence was measured. Switch 1.0 alone had low fluorescence, while Switch 1.0 with trigger had high fluorescence (Figure 1). Switch 1.0 was then tested with thrombin induction, at varying concentrations of thrombin. Trigger and aptamer were pre-incubated to allow them to hybridize and prevent unbound trigger from binding the toehold region and then added to the in vitro translation kits, switch DNA, and thrombin. High expression was seen for all combinations, including switch with trigger and aptamer with 0 uM thrombin, with the one exception being 25 uM thrombin (Figure 2). As this was the opposite of the desired result, Switch 1.0 was then tested without any aptamer. This experiment showed that high concentrations of thrombin could be inhibiting the switch (Figure 3). This inspired the Switch 2.0 design, where the protein binds directly to the switch in a different way, causing the hairpin to unfold and the switch to be turned on. Switch 2.0 was tested with thrombin induction at varying concentrations of thrombin and fluorescence increased with increasing thrombin (Figure 4).

Figure 1: Toehold Switches with and without triggers
Switch 1.0 was adapted for thrombin detection with a GFP reporter and induced by the corresponding DNA trigger, showing over 100,000 RFU, while the uninduced switch showed minimal background.


Figure 2: Thrombin Titration (switch 1.0 + trigger + aptamer + thrombin), 9/7/15 results
The positive control, consisting of Switch 1.0 with trigger had high expression. The negative control, consisting of Switch 1.0 with pre-incubated trigger and aptamer had high expression. Switch 1.0 with trigger, aptamer, and 0.2 uM-5.0 uM thrombin had high expression, and 25.0 uM had low expression, the opposite of the intended design. This experiment was repeated several times with the same result.


Figure 3: Positive Control (switch + trigger, no aptamer) 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 adapted for thrombin detection and induced with 0.2-5uM thrombin had increasing expression with increasing thrombin concentrations. Negative control, consisting of Switch 2.0 with no thrombin, had minimal expression. Blank, consisting of water, had minimal fluorescence. 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.


Discussion
Two different switch designs were tested with thrombin and Switch 2.0 responded successfully, even at the lowest concentration of thrombin tested.

Switch 1.0 was successfully induced by the DNA trigger; however, 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 the aptamer and trigger are unbound under the experimental conditions (no added salt, 37 °C), allowing high background. Additionally, fluorescence is low at high concentrations of thrombin, implying that thrombin is inhibiting translation of GFP. One possibility is that thrombin inhibits switch activation by binding to the toehold region, which is the RNA version of the DNA aptamer. This inspired the successful Switch 2.0 design.

Switch 2.0 was successfully induced by thrombin. Switch 2.0 showed an increase in GFP expression as thrombin protein concentration increased and responded to the lowest concentration tested, 0.2uM. The thrombin version of Switch 2.0 shows much potential, and it seems promising that adapting the switch for other proteins could yield similar results.

Altogether, 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 only show one iteration of each switch. Thus, Switch 2.0 was successful with zero optimization of the design, and it is likely that performance could be further improved. Binding strength of different parts of each switch (toehold to protein, hairpin) could be optimized to adapt Switch 2.0 for a wide range of proteins, while maintaining high reporter expression and low background.

Future Directions
- Adapt, test, and determine limit of detection of Switch 2.0 for a variety of proteins
- Optimize to increase reporter expression for Switch 2.0 and reduce background
- Test specificity using biological sample
- Demonstrate on paper using a colorimetric reporter


References

  1. Pardee, K., Green, A., Ferrante, T., Cameron, D., Daleykeyser, A., Yin, P., & Collins, J. (n.d.). Paper-Based Synthetic Gene Networks. Cell, 940-954.