Difference between revisions of "Team:Pitt/Description"

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<br/><br /><span style="display: none;">Cell-free extracts are essentially composed of lysed cells. In fact, it was in cell-free extracts that the genetic code was discovered, as it allowed for easy control of translation. Since <span class="edit">YEAR</span>, significant advancements have been made in this technology, providing extremely fine control over both transcription and translation. In its simplest form, a cell-free extract made from <i>E. coli</i> will contain all the components of the prokaryotic cell, except for the cell wall, the membrane, and the chromosomal DNA attached to the membrane. While this extract will be able to both transcribe DNA, and translate the resulting RNA, both processes occur at very low rates. During cell growth, a cell will produce and reuse a variety of small molecules, including but not limited to nucleotide triphosphates (NTPs) and amino acids. Both of these are essential to <i>in vitro</i> transcription and translation. However, once the chromosomal DNA is removed, the extract retains very little of its ability to create small molecules and create high energy molecules from energy sources such as glucose. With this realization, a number of improvements to the cell-free extract were made, by adding the necessary components to the final reaction mixture. The amino acids and NTPs are added in great excess, which not only provides the starting materials for RNA and protein synthesis, but also increases the thermodynamic drive for the synthesis of macromolecules. In addition, creatine kinase is added along with creatine phosphate as a way of regenerating high energy molecules such as ATP from ADP. After these modifications to the cell-free extract protocol, there was about a (number) increase in the rate of production of protein from extracts. However, it was soon realized that the majority of ribosomes were still bound to mRNAs from the lysed cell, some of which stalled during translation, which diverted some of the energy from producing the protein of interest to producing short peptides and other proteins. To counteract this source of contamination, an additional incubation step was added, followed by a dialysis step to free as many of the ribosomes as possible. Th final modifications to the currently accepted cell-free extract protocol were made in 2005<span class="edit">REF</span>, and our group used a similar method. However, the goal of most groups is to produce as much protein as possible, for which they require an extract as free of other proteins as possible. For the purpose of this project, proteins that regulate transcription are needed in the cell extract. One way of achieving this is by purifying the protein from a cell extract, then adding to the final reaction mixture. However, a simpler approach is to induce the protein of interest directly in the cells from which the extract is made. By choosing this approach, the number of steps required for the production of our "sensor extracts" (SE) was greatly reduced. Currently, the entire production process requires 3 days, and the final cost of a single sensor is <span class="edit">MATH</span>. To see more, click <a href="2015.igem.org/Team:Pitt/Protocols">here</a>. For sensor extracts, it is typical to include proteins that allow for the modulation of transcriptional activation in the presence of an analyte, while the final reaction mix contains a DNA construct that contains the appropriate promoter followed by a reporter gene (typically a fluorescent protein such as GFP). <button class="minimizer">Click to read about other aspects of the project...</button><span>
 
<br/><br /><span style="display: none;">Cell-free extracts are essentially composed of lysed cells. In fact, it was in cell-free extracts that the genetic code was discovered, as it allowed for easy control of translation. Since <span class="edit">YEAR</span>, significant advancements have been made in this technology, providing extremely fine control over both transcription and translation. In its simplest form, a cell-free extract made from <i>E. coli</i> will contain all the components of the prokaryotic cell, except for the cell wall, the membrane, and the chromosomal DNA attached to the membrane. While this extract will be able to both transcribe DNA, and translate the resulting RNA, both processes occur at very low rates. During cell growth, a cell will produce and reuse a variety of small molecules, including but not limited to nucleotide triphosphates (NTPs) and amino acids. Both of these are essential to <i>in vitro</i> transcription and translation. However, once the chromosomal DNA is removed, the extract retains very little of its ability to create small molecules and create high energy molecules from energy sources such as glucose. With this realization, a number of improvements to the cell-free extract were made, by adding the necessary components to the final reaction mixture. The amino acids and NTPs are added in great excess, which not only provides the starting materials for RNA and protein synthesis, but also increases the thermodynamic drive for the synthesis of macromolecules. In addition, creatine kinase is added along with creatine phosphate as a way of regenerating high energy molecules such as ATP from ADP. After these modifications to the cell-free extract protocol, there was about a (number) increase in the rate of production of protein from extracts. However, it was soon realized that the majority of ribosomes were still bound to mRNAs from the lysed cell, some of which stalled during translation, which diverted some of the energy from producing the protein of interest to producing short peptides and other proteins. To counteract this source of contamination, an additional incubation step was added, followed by a dialysis step to free as many of the ribosomes as possible. Th final modifications to the currently accepted cell-free extract protocol were made in 2005<span class="edit">REF</span>, and our group used a similar method. However, the goal of most groups is to produce as much protein as possible, for which they require an extract as free of other proteins as possible. For the purpose of this project, proteins that regulate transcription are needed in the cell extract. One way of achieving this is by purifying the protein from a cell extract, then adding to the final reaction mixture. However, a simpler approach is to induce the protein of interest directly in the cells from which the extract is made. By choosing this approach, the number of steps required for the production of our "sensor extracts" (SE) was greatly reduced. Currently, the entire production process requires 3 days, and the final cost of a single sensor is <span class="edit">MATH</span>. To see more, click <a href="2015.igem.org/Team:Pitt/Protocols">here</a>. For sensor extracts, it is typical to include proteins that allow for the modulation of transcriptional activation in the presence of an analyte, while the final reaction mix contains a DNA construct that contains the appropriate promoter followed by a reporter gene (typically a fluorescent protein such as GFP). <button class="minimizer">Click to read about other aspects of the project...</button><span>
 
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     <td colspan="3" class="td50"><h4>Clear Responses: Amplification and Quenching</h4><br/>One 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. <button class="expander">Click to see more...</button><br /> <br /> <br /> <span style="display: none;">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. {picture} 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. To learn more about this sub-project, click here.<button class="minimizer">return</button></span>
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     <td colspan="3" class="td50"><h4>Clear Responses: Amplification and Quenching</h4><br/>One 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. <button class="expander">Click to read more...</button><br /> <br /> <br /> <span style="display: none;">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. <span class="edit">PIC</span> 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. To learn more about this sub-project, click <a href="2015.igem.org/Team:Pitt/Amplification/Project">here</a>.<button class="minimizer">Click to read about other aspects of the project...</button></span>
 
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<tr><td colspan="2" class="td33"><h4>Estrogen Sensor</h4><br/>While it has been shown that transcription in cell-free extracts can rely on RNA polymerases sensitive to small molecules, our team decided to test the viability of using such polymerases in our cheap, home-made sensor extracts, rather than in expensive, commercially available extracts. In doing so, we also characterized a part from CMU's iGEM team, the estrogen-sensitive T7 RNA polymerase<button class="expander">Click to see more...</button><span style="display: none;">blah blahblah <button class="minimizer">return</button></span></td>
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<tr><td colspan="2" class="td33"><h4>Estrogen Sensor</h4><br/>While it has been shown that transcription in cell-free extracts can rely on RNA polymerases sensitive to small molecules<span class="edit">REF</span>, our team decided to test the viability of using such polymerases in our cheap, home-made sensor extracts, rather than in expensive, commercially available extracts. In doing so, we used a part from CMU's iGEM team, the estrogen-sensitive T7 RNA polymerase.<br/><button class="expander">Click to read more...</button><span style="display: none;"><br/><br/>This subproject was the first and simplest application of our sensor extracts. Since CMU's iGEM team had been working on an estrogen-sensitive mutant of T7 RNA Polymerase, we decided to use the construct in a cell-free context. Sensors that detect estrogen quickly could be used in a variety of contexts, including but not limited to quantifying estrogen in blood, and detecting estrogen-contaminated water. <br/><button class="minimizer">Click to read about other aspects of the project...</button></span></td>
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<td colspan="2" class="td33"><h4>Protease Sensor</h4><br/>The second sensing system we have designed relies on transcriptional repressors. By creating a synthetic repressor that gets cleaved by a specific protease, the extract we create will be sensitive to the protease. This can be used to detect breast and colorectal cancer biomarkers such as MMP-2 and MMP-9 in patients' urine.<button class="expander">Click to see more...</button><span style="display: none;">blah blahblah <button class="minimizer">return</button></span></td>
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<td colspan="2" class="td33"><h4>Protease Sensor</h4><br/>The second sensing system we have designed relies on transcriptional repressors. By creating a synthetic repressor that gets cleaved by a specific protease, the extract we create will be sensitive to the protease. This can be used to detect breast and colorectal cancer biomarkers such as MMP-2 and MMP-9 in patients' urine.<button class="expander">Click to see more...</button><span style="display: none;"><button class="minimizer">Click to read about other aspects of the project...</button></span></td>
 
<td colspan="2" class="td33"><h4>Three-Hybrid Versatile Sensor</h4><br/>This is a cool thing<br/><button class="expander">Click to see more...</button><span style="display: none;">blah blahblah <button class="minimizer">return</button></span></td></tr>
 
<td colspan="2" class="td33"><h4>Three-Hybrid Versatile Sensor</h4><br/>This is a cool thing<br/><button class="expander">Click to see more...</button><span style="display: none;">blah blahblah <button class="minimizer">return</button></span></td></tr>
 
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Revision as of 19:42, 18 September 2015

Cheap biosensors based on cell-free extracts

Pitt's 2015 iGEM team investigated the possibility of using cell-free extracts as a method of detecting extremely small concentrations of biomolecules. Furthermore, these sensors were tested after being freeze-dried on paper, to test the feasibility of mass-producing and distributing cheap sensors worldwide. While the ideas tested in this project can be applied to sense an almost unlimited number of biomolecules, we focused on three major sensors.

Project Idea and Motivation


The ability to detect small concentrations of molecules accurately without the use of lab equipment is a huge step in creating portable sensing devices. While many extremely sensitive methods have been developed, there are very few that are amenable to work in the field, or at home as a consumer product. This project focuses on creating methods that allow for simple and quick detection of biomolecules without the use of laboratory instruments.

Recently, it was shown that freeze-drying cell extracts on paper has little effect on the ability of the cell extract to perform in vitro transcription and translation.REF Since paper strips are simple to transport and store, we chose this media to create our sensors. This method has the added advantage of utilizing the natural amplification of transcription and translation to visualize incredibly small concentrations without the use of complicated instrumentation. Furthermore, results can be available in as little as an hourREF, which is significantly quicker than most routine lab tests. Based on these ideas, out final goal was to create a sensor that would function similarly to the at-home pregnancy test. While this project focuses on a proof-of-concept, the system is designed to be extremely versatile, so three different sensors were conceptualized.

Cell-free Extracts


The use of cell-free extracts for sensors allows us to solve several problems at once. First of all, by using the natural amplification of both transcription and translation in vitro, extremely small amounts of analyte can be detected. Furthermore, it was recently shown that these cell extracts retain their function when freeze-dried on paper, which allows for easy transport of the completed sensor.REF


Cell-free extracts are essentially composed of lysed cells. In fact, it was in cell-free extracts that the genetic code was discovered, as it allowed for easy control of translation. Since YEAR, significant advancements have been made in this technology, providing extremely fine control over both transcription and translation. In its simplest form, a cell-free extract made from E. coli will contain all the components of the prokaryotic cell, except for the cell wall, the membrane, and the chromosomal DNA attached to the membrane. While this extract will be able to both transcribe DNA, and translate the resulting RNA, both processes occur at very low rates. During cell growth, a cell will produce and reuse a variety of small molecules, including but not limited to nucleotide triphosphates (NTPs) and amino acids. Both of these are essential to in vitro transcription and translation. However, once the chromosomal DNA is removed, the extract retains very little of its ability to create small molecules and create high energy molecules from energy sources such as glucose. With this realization, a number of improvements to the cell-free extract were made, by adding the necessary components to the final reaction mixture. The amino acids and NTPs are added in great excess, which not only provides the starting materials for RNA and protein synthesis, but also increases the thermodynamic drive for the synthesis of macromolecules. In addition, creatine kinase is added along with creatine phosphate as a way of regenerating high energy molecules such as ATP from ADP. After these modifications to the cell-free extract protocol, there was about a (number) increase in the rate of production of protein from extracts. However, it was soon realized that the majority of ribosomes were still bound to mRNAs from the lysed cell, some of which stalled during translation, which diverted some of the energy from producing the protein of interest to producing short peptides and other proteins. To counteract this source of contamination, an additional incubation step was added, followed by a dialysis step to free as many of the ribosomes as possible. Th final modifications to the currently accepted cell-free extract protocol were made in 2005REF, and our group used a similar method. However, the goal of most groups is to produce as much protein as possible, for which they require an extract as free of other proteins as possible. For the purpose of this project, proteins that regulate transcription are needed in the cell extract. One way of achieving this is by purifying the protein from a cell extract, then adding to the final reaction mixture. However, a simpler approach is to induce the protein of interest directly in the cells from which the extract is made. By choosing this approach, the number of steps required for the production of our "sensor extracts" (SE) was greatly reduced. Currently, the entire production process requires 3 days, and the final cost of a single sensor is MATH. To see more, click here. For sensor extracts, it is typical to include proteins that allow for the modulation of transcriptional activation in the presence of an analyte, while the final reaction mix contains a DNA construct that contains the appropriate promoter followed by a reporter gene (typically a fluorescent protein such as GFP).

Clear Responses: Amplification and Quenching


One 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. PIC 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. To learn more about this sub-project, click here.

Estrogen Sensor


While it has been shown that transcription in cell-free extracts can rely on RNA polymerases sensitive to small moleculesREF, our team decided to test the viability of using such polymerases in our cheap, home-made sensor extracts, rather than in expensive, commercially available extracts. In doing so, we used a part from CMU's iGEM team, the estrogen-sensitive T7 RNA polymerase.


This subproject was the first and simplest application of our sensor extracts. Since CMU's iGEM team had been working on an estrogen-sensitive mutant of T7 RNA Polymerase, we decided to use the construct in a cell-free context. Sensors that detect estrogen quickly could be used in a variety of contexts, including but not limited to quantifying estrogen in blood, and detecting estrogen-contaminated water.

Protease Sensor


The second sensing system we have designed relies on transcriptional repressors. By creating a synthetic repressor that gets cleaved by a specific protease, the extract we create will be sensitive to the protease. This can be used to detect breast and colorectal cancer biomarkers such as MMP-2 and MMP-9 in patients' urine.

Three-Hybrid Versatile Sensor


This is a cool thing
blah blahblah