Difference between revisions of "Team:Pitt/Amplification/Project"
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<div class="color"><h4>Project Background</h4>TOne of the key aspects of creating a reliable and useful sensor is having a clear yes/no response. A good example of this is the at-home pregnancy test, where 2 lines indicates pregnancy, and 1 line indicates no pregnancy. While it is impossible to remove <i>all</i> outliers, we have been working on a system that will amplify positive signals, while quenching noise. A huge advantage of using cell extracts is the possibility to create synthetic gene circuits that can modulate the response of the sensor. For this project, we created a rather simple circuit that accounts for noise, while amplifying positive signals. All three sensors that we are building rely on transcriptional activation. Thus, provided that each circuit outputs the same protein at the end of one transcription/translation cycle, the same modular amplification and quenching circuit can be applied to all 3. <br/><img style="width:75%" src="https://static.igem.org/mediawiki/2015/b/bc/Pitt3.png"/><br/> As shown above, we have chosen the output from the first system to be the T3 phage RNA polymerase. By using this polymerase (which has a different promoter than any of the systems we have designed), we can amplify the signal several times through a positive feedback construct that produces more T3 RNAP in the presence of T3 RNAP. Finally, by including a DNA construct that produces a reporter (typically GFP for simple measurement by a fluorimeter), the result of the amplification can be seen. Unfortunately, this circuit also amplifies noise extremely well, which typically occurs due to leaky expression of the sensor promoter. To counteract this, a quencher was designed that binds to T3 RNAP and blocks the polymerase from transcribing, and ultimately from amplifying the noise. The modular design of the circuit allows for careful fine-tuning for each system. By increasing the amount of amplification construct, lower levels of detection can potentially be achieved, while increasing the amount of quencher can account for a larger amount of leaky transcription.</div> | <div class="color"><h4>Project Background</h4>TOne of the key aspects of creating a reliable and useful sensor is having a clear yes/no response. A good example of this is the at-home pregnancy test, where 2 lines indicates pregnancy, and 1 line indicates no pregnancy. While it is impossible to remove <i>all</i> outliers, we have been working on a system that will amplify positive signals, while quenching noise. A huge advantage of using cell extracts is the possibility to create synthetic gene circuits that can modulate the response of the sensor. For this project, we created a rather simple circuit that accounts for noise, while amplifying positive signals. All three sensors that we are building rely on transcriptional activation. Thus, provided that each circuit outputs the same protein at the end of one transcription/translation cycle, the same modular amplification and quenching circuit can be applied to all 3. <br/><img style="width:75%" src="https://static.igem.org/mediawiki/2015/b/bc/Pitt3.png"/><br/> As shown above, we have chosen the output from the first system to be the T3 phage RNA polymerase. By using this polymerase (which has a different promoter than any of the systems we have designed), we can amplify the signal several times through a positive feedback construct that produces more T3 RNAP in the presence of T3 RNAP. Finally, by including a DNA construct that produces a reporter (typically GFP for simple measurement by a fluorimeter), the result of the amplification can be seen. Unfortunately, this circuit also amplifies noise extremely well, which typically occurs due to leaky expression of the sensor promoter. To counteract this, a quencher was designed that binds to T3 RNAP and blocks the polymerase from transcribing, and ultimately from amplifying the noise. The modular design of the circuit allows for careful fine-tuning for each system. By increasing the amount of amplification construct, lower levels of detection can potentially be achieved, while increasing the amount of quencher can account for a larger amount of leaky transcription.</div> | ||
| − | <div class="color"><h4>Project State</h4>Currently, we are cloning the amplification loop constructs using Biobrick assembly. However, we have had reasonable success with our decoy. Since we have been unable to clone any constructs with T3 RNAP, we used T7 RNA Polymerase driven expression of GFP. By varying the concentration of decoy, we were able to control the level of expression of GFP, as seen in the graph below.</div> | + | <div class="color"><h4>Project State</h4>Currently, we are cloning the amplification loop constructs using Biobrick assembly. However, we have had reasonable success with our decoy. Since we have been unable to clone any constructs with T3 RNAP, we used T7 RNA Polymerase driven expression of GFP. By varying the concentration of decoy, we were able to control the level of expression of GFP, as seen in the graph below. <br/> <img style="width:65%;" src="https://static.igem.org/mediawiki/2015/b/b7/PittDecoy.PNG"/></div> |
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Latest revision as of 02:32, 19 September 2015
Amplification and Quenching Circuit
Project Background
TOne of the key aspects of creating a reliable and useful sensor is having a clear yes/no response. A good example of this is the at-home pregnancy test, where 2 lines indicates pregnancy, and 1 line indicates no pregnancy. While it is impossible to remove all outliers, we have been working on a system that will amplify positive signals, while quenching noise. A huge advantage of using cell extracts is the possibility to create synthetic gene circuits that can modulate the response of the sensor. For this project, we created a rather simple circuit that accounts for noise, while amplifying positive signals. All three sensors that we are building rely on transcriptional activation. Thus, provided that each circuit outputs the same protein at the end of one transcription/translation cycle, the same modular amplification and quenching circuit can be applied to all 3.
As shown above, we have chosen the output from the first system to be the T3 phage RNA polymerase. By using this polymerase (which has a different promoter than any of the systems we have designed), we can amplify the signal several times through a positive feedback construct that produces more T3 RNAP in the presence of T3 RNAP. Finally, by including a DNA construct that produces a reporter (typically GFP for simple measurement by a fluorimeter), the result of the amplification can be seen. Unfortunately, this circuit also amplifies noise extremely well, which typically occurs due to leaky expression of the sensor promoter. To counteract this, a quencher was designed that binds to T3 RNAP and blocks the polymerase from transcribing, and ultimately from amplifying the noise. The modular design of the circuit allows for careful fine-tuning for each system. By increasing the amount of amplification construct, lower levels of detection can potentially be achieved, while increasing the amount of quencher can account for a larger amount of leaky transcription.
Project State
Currently, we are cloning the amplification loop constructs using Biobrick assembly. However, we have had reasonable success with our decoy. Since we have been unable to clone any constructs with T3 RNAP, we used T7 RNA Polymerase driven expression of GFP. By varying the concentration of decoy, we were able to control the level of expression of GFP, as seen in the graph below.