Team:Caltech/Project

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<b><font size=+1>Overall Project Summary</font></b>
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<b><font size=+1>Project Overview</font></b>
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<a href = "Project/Experiments">The Experiments</a>
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<a href = "Project/Results">Our Results</a>
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<h3>Background and Motivation</h3>
<h3>Background and Motivation</h3>
<p>One of synthetic biology's chief promises is the potential to construct artificial, self-regulatory genetic circuits. While intracellular genetic regulation, as exemplified by such model systems as the repressilator, is fairly well documented and explored, intercellular regulation, mediated by cell-to-cell signaling, remains less well characterized and explored. This year, the Caltech 2014 iGEM team focused on the implementation of non-endogenous bacterial quorum-sensing systems in <i>E. coli</i>, in hopes that successful transfer of these systems into E. coli will promote enhanced prototyping of intercellular—in addition to intracellular—signaling and regulation in synthetic biology’s favorite model organism.</p>
<p>One of synthetic biology's chief promises is the potential to construct artificial, self-regulatory genetic circuits. While intracellular genetic regulation, as exemplified by such model systems as the repressilator, is fairly well documented and explored, intercellular regulation, mediated by cell-to-cell signaling, remains less well characterized and explored. This year, the Caltech 2014 iGEM team focused on the implementation of non-endogenous bacterial quorum-sensing systems in <i>E. coli</i>, in hopes that successful transfer of these systems into E. coli will promote enhanced prototyping of intercellular—in addition to intracellular—signaling and regulation in synthetic biology’s favorite model organism.</p>
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<p> The original motivation for this project was to create a model system that will resemble the natural hormone regulatory systems of the body. Typically, these regulatory systems are responsible for maintaining normal functioning of vital human bodily processes including stabilizing blood glucose and cortisol levels. Breakdowns of these regulatory systems, as exemplified in diseases such as diabetes, hyperthyroidism, and Cushing's disease, represent many of the major obstacles facing modern medicine. The original intent of the project was to create a model system analogous to one of these regulatory systems in E. coli, showing one plausible framework to serve as a scaffold for further research that could one day be used to present a solution to one or more of these diseases.</p>
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<p> The original motivation for this project was to create a model system that will resemble the natural hormone regulatory systems of the body. Typically, these regulatory systems are responsible for maintaining normal functioning of vital human bodily processes including stabilizing blood glucose and cortisol levels. Breakdowns of these regulatory systems, as exemplified in diseases such as diabetes (<b>Figure 1</b>), hyperthyroidism, and Cushing's disease, represent many of the major obstacles facing modern medicine. The original intent of the project was to create a model system analogous to one of these regulatory systems in E. coli, showing one plausible framework to serve as a scaffold for further research that could one day be used to present a solution to one or more of these diseases.</p>
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<h3>Our Original Project Design</h3>
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<p> Originally, we had planned to construct an autoregulatory genetic circuit to serve as a model system for the synthesis and regulation of a biological compound outside the cell (see Figure 1). In this system, a protein—comX—was to be synthesized and exported by the cell. That protein was then intended to bind a receptor on the cell membrane, activating a negative feedback loop to halt synthesis and export of the original protein, resulting in stable concentrations of the original protein in the long run. Successful construction and characterization of this system would have offered proof of concept that, in the future, many different molecules can be synthesized and regulated at stable levels using the same overall architecture. Given the large number of human diseases caused by inability of the body to self-regulate levels of an essential chemical compound (e.g. insulin in diabetes, LDL in hyperlipidemia, etc.), there is a strong push to create such types of artificial gene circuits in hopes of finally finding long-term solutions to these diseases.</p>
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<table width=70%><tr><td><b>Figure 1. Our Initial Circuit Design.</b> In the design of the original circuit, we attempted to implement a negative feedback loop across a cell membrane by introducing proteins from the comQXPA quorum sensing system native to Bacillus subtilis. In this system, comX, the signaling peptide would be produced and exported by comQ, a membrane protein. comX then would then bind comP, a membrane receptor, activating comA to activate transcription of a repressor protein that would repress expression of comX. mNeonGreen was originally planned to be attached to comX for ease of experimental detection.</td></tr></table>
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<table width=70%><tr><td><b>Figure 1. Proper Glucose Regulation. </b>In a normal human, insulin is produced in the beta cells of the pancreas, post-translationally modified, and then shipped out of the cell via exocytosis. It is then picked up by tyrosine kinase receptors on muscle cells, activating a signal transduction pathway ultimately leading to glucose uptake from the blood via endocytosis. In a patient with diabetes, depending on the type of diabetes, some aspect of this pathway stops functioning.</td></tr></table>
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<p>During design of this circuit, however, it was soon found that the comQXPA quorum sensing system native to B. subtilis that we had hoped to implement in the system to regulate the negative feedback loop would not be able to be imported into E. coli due to differences between the two species’ cell membrane composition [1]. Furthermore, an extensive literature search demonstrated that there exists very little extant research, in general, into the viability of implementing quorum sensing systems from one species of bacteria into non-native environments.</p>
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<h3>Project Design</h3>
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<p>Seeing this deficiency, we have modified our system to focus more specifically on testing cell-to-cell signaling. The new circuit design features 2 separate cells splitting the components of the quorum sensing system between the two different cells such that “cell 1” will create a peptide signaling ligand that is used to signal exclusively “cell 2” expressing the receptor for that ligand on its surface (see Figure 2), activating an easily detectable response—expression of GFP—in the second cell.</p>
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<p> Originally, we had planned to construct an autoregulatory genetic circuit to serve as a model system for the synthesis and regulation of a biological compound outside the cell (see <b>Figure 2</b>). This model system would accomplish this by incorporating components from bacterial quorum sensing systems—specifically, we decided on the comQXPA system natively found in <i>Bacillus subtilis</i>. The protein comX was to be synthesized and exported by the cell. Then it was intended to bind a receptor on the cell membrane, activating a negative feedback loop to halt synthesis and export of the original protein, resulting in stable concentrations of the original protein in the long run. Successful construction and characterization of this system would have offered proof of concept that, in the future, many different molecules can be synthesized and regulated at stable levels using the same overall architecture. Given the large number of human diseases caused by inability of the body to self-regulate levels of an essential chemical compound (e.g. insulin in diabetes, LDL in hyperlipidemia, etc.), there is a strong push to create such types of artificial gene circuits in hopes of finally finding long-term solutions to these diseases.</p>
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<table width=70%><tr><td><b>Figure 2. Circuit Design Modified for Quorum Sensing System Testing.</b> The new circuit design splits the quorum sensing systems’ parts into 2 cells (one with the ligand and necessary export and modification machinery and one with the histidine kinase receptor and response regulator). Activation of ligand-creation in cell 1 via IPTG addition should result in fluorescence in cell 2.</td></tr></table>
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<table width=70%><tr><td><b>Figure 2. Our Initial Circuit Design.</b> In the design of the original circuit, we attempted to implement a negative feedback loop across a cell membrane by introducing proteins from the comQXPA quorum sensing system native to Bacillus subtilis. In this system, comX, the signaling peptide would be produced and exported by comQ, a membrane protein. comX then would then bind comP, a membrane receptor, activating comA to activate transcription of a repressor protein that would repress expression of comX. mNeonGreen was originally planned to be attached to comX for ease of experimental detection.</td></tr></table>
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<p>During design of this circuit, however, it was soon found that the comQXPA quorum sensing system native to B. subtilis that we had hoped to implement in the system to regulate the negative feedback loop would not be able to be imported into E. coli due to differences between the two species’ cell membrane composition [1]. Furthermore, an extensive literature search demonstrated that there exists very little extant research, in general, into the viability of implementing quorum sensing systems from one species of bacteria into non-native environments.</p>
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<h3>Quorum Sensing Systems Investigated</h3>
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<p>Seeing this deficiency, we have modified our system to focus more specifically on testing cell-to-cell signaling. The new circuit design features 2 separate cells splitting the components of the quorum sensing system between the two different cells such that “cell 1” will create a peptide signaling ligand that is used to signal exclusively “cell 2” expressing the receptor for that ligand on its surface (see <b>Figure 3</b>), activating an easily detectable response—expression of GFP—in the second cell.</p>
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<i>agrBDCA system</i>
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<p>The agrBDCA quorum sensing system is a QS system native to Streptococcus aureus and is composed of 4 proteins: agrB, agrD, agrC, and agrA. agrB is a membrane protein involved in the export and modification/processing of agrD, the precursor peptide that is modified into the signaling peptide. Once exported out of the cell and fully modified, the ligand then binds to agrC, a histidine kinase receptor. Upon binding the ligand, agrC then phosphorylates the response regulator agrA, which acts as a transcriptional activator for the P2 promoter, which we have placed in front of the reporter GFP [2]. Figure 3 illustrates the action of this system on itself. In the endogenous system, the P2 promoter is actually found before the entire agrBDCA operon, so that the entire system forms a self-inducing, positive feedback loop. In its context as a quorum sensing system regulating virulence factors, it makes perfect sense as a system that will "push itself forward" and progress, once <i>S. aureus</i> reach a critical density. However, in our system, we have segregated the components of the system such that, in the signaling cell, we are controlling ligand production via a pTet promoter. Only in the receiving cell are we expressing the P2 promoter to activate expression of the GFP reporter protein.</p>
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<table width=70%><tr><td><b>Figure 3. agrBDCA Quorum Sensing System.</b> In the agr quorum sensing system, agrB is a membrane protein that modifies agrD, the precursor peptide, into its mature form before shipping it out of the cell. From there, the mature ligand binds agrC, the histidine kinase receptor, causing it to phosphorylate agrA, the response regulator. The activated agrA then binds the P2 and P3 promoters as an inducer, which further promotes expression of the quorum sensing system and mediates additional gene regulation (RNAIII) in the native setup shown here.</td></tr></table>
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<table width=70%><tr><td><b>Figure 3. Circuit Design Modified for Quorum Sensing System Testing.</b> The new circuit design splits the quorum sensing systems’ parts into 2 cells (one with the ligand and necessary export and modification machinery and one with the histidine kinase receptor and response regulator). Activation of ligand-creation in cell 1 via IPTG addition should result in fluorescence in cell 2. Note as well the resemblance between this system and that for insulin-mediated glucose regulation shown in <b>Figure 1</b>.</td></tr></table>
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<i>lamBDCA System</i>
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<p>Additionally, one of the focuses of our project was on the implementation of combinatorial promoters to increase the functionality  of the pResponse promoter. This component of the project originated from our original circuit design, in which a combinatorial promoter would have been necessary for correct functioning of the circuit (refer back to <b>Figure 2</b>), but we continued characterization of different combinatorial promoters during the summer, even after we had abandoned the original circuit design, in hopes that it could add functionality to our final circuit even though it was no longer necessary.</p>
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<p>Additionally, we investigated the lamBDCA QS system, a quorum sensing system native to <i>Lactobacillus plantarum</i>. In its native environment, the lamBDCA system regulates adherence of <i>L. plantarum</i> to surfaces and cell morphology. Of note is that it shares significant homology with the agrBDCA quorum-sensing system in <i>S. aureus</i>: the components of the lamBDCA system mirror those of agrBDCA exactly (i.e. lamA is the response regulator, lamC is the histidine kinase receptor, lamB is the membrane protein, and lamD is the signaling ligand’s precursor peptide). [3]</p>
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The Caltech iGEM team plans to use the principles and methods of synthetic biology to create a model system that can offer a framework for future researchers to study the dynamics of diseases like diabetes. The gene circuit has been designed so that "Cell 1" will produce a signaling peptide upon induction with aTc. This signaling peptide will then attach to the receptor of "Cell 2," triggering a very simple signal transduction pathway leading to expression of some target gene that, for our testing purposes, will be the reporter protein GFP. The quorum sensing systems being imported into the <i>E. coli</i> cells come from Gram-positive species of bacteria, which minimizes the possibility that their activity will interfere with or be interfered with by <i>E. coli's</i> endogenous signaling and reception systems but also introduces the challenge of trying to implement the relevant membrane proteins in an environment that significantly differs from their native environments (owing to the differences in membrane composition between Gram-positive and Gram-negative bacteria).</p>
 
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Our project, however, is not without precedent: we will be building off of TU Delft's team project in 2013, which managed successfully to implement the signal reception components of the agrBCDA quorum sensing system in <i>E. coli</i>. </p>
 
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Latest revision as of 21:38, 17 October 2014



Home Team Official Team Profile Project Parts TXTL Promoter Characterization Notebook Safety Attributions
Project Description
Project Overview

Project Details

Materials and Methods

The Experiments

Our Results

Conclusions

References

Background and Motivation

One of synthetic biology's chief promises is the potential to construct artificial, self-regulatory genetic circuits. While intracellular genetic regulation, as exemplified by such model systems as the repressilator, is fairly well documented and explored, intercellular regulation, mediated by cell-to-cell signaling, remains less well characterized and explored. This year, the Caltech 2014 iGEM team focused on the implementation of non-endogenous bacterial quorum-sensing systems in E. coli, in hopes that successful transfer of these systems into E. coli will promote enhanced prototyping of intercellular—in addition to intracellular—signaling and regulation in synthetic biology’s favorite model organism.

The original motivation for this project was to create a model system that will resemble the natural hormone regulatory systems of the body. Typically, these regulatory systems are responsible for maintaining normal functioning of vital human bodily processes including stabilizing blood glucose and cortisol levels. Breakdowns of these regulatory systems, as exemplified in diseases such as diabetes (Figure 1), hyperthyroidism, and Cushing's disease, represent many of the major obstacles facing modern medicine. The original intent of the project was to create a model system analogous to one of these regulatory systems in E. coli, showing one plausible framework to serve as a scaffold for further research that could one day be used to present a solution to one or more of these diseases.



Figure 1. Proper Glucose Regulation. In a normal human, insulin is produced in the beta cells of the pancreas, post-translationally modified, and then shipped out of the cell via exocytosis. It is then picked up by tyrosine kinase receptors on muscle cells, activating a signal transduction pathway ultimately leading to glucose uptake from the blood via endocytosis. In a patient with diabetes, depending on the type of diabetes, some aspect of this pathway stops functioning.

Project Design

Originally, we had planned to construct an autoregulatory genetic circuit to serve as a model system for the synthesis and regulation of a biological compound outside the cell (see Figure 2). This model system would accomplish this by incorporating components from bacterial quorum sensing systems—specifically, we decided on the comQXPA system natively found in Bacillus subtilis. The protein comX was to be synthesized and exported by the cell. Then it was intended to bind a receptor on the cell membrane, activating a negative feedback loop to halt synthesis and export of the original protein, resulting in stable concentrations of the original protein in the long run. Successful construction and characterization of this system would have offered proof of concept that, in the future, many different molecules can be synthesized and regulated at stable levels using the same overall architecture. Given the large number of human diseases caused by inability of the body to self-regulate levels of an essential chemical compound (e.g. insulin in diabetes, LDL in hyperlipidemia, etc.), there is a strong push to create such types of artificial gene circuits in hopes of finally finding long-term solutions to these diseases.



Figure 2. Our Initial Circuit Design. In the design of the original circuit, we attempted to implement a negative feedback loop across a cell membrane by introducing proteins from the comQXPA quorum sensing system native to Bacillus subtilis. In this system, comX, the signaling peptide would be produced and exported by comQ, a membrane protein. comX then would then bind comP, a membrane receptor, activating comA to activate transcription of a repressor protein that would repress expression of comX. mNeonGreen was originally planned to be attached to comX for ease of experimental detection.

During design of this circuit, however, it was soon found that the comQXPA quorum sensing system native to B. subtilis that we had hoped to implement in the system to regulate the negative feedback loop would not be able to be imported into E. coli due to differences between the two species’ cell membrane composition [1]. Furthermore, an extensive literature search demonstrated that there exists very little extant research, in general, into the viability of implementing quorum sensing systems from one species of bacteria into non-native environments.

Seeing this deficiency, we have modified our system to focus more specifically on testing cell-to-cell signaling. The new circuit design features 2 separate cells splitting the components of the quorum sensing system between the two different cells such that “cell 1” will create a peptide signaling ligand that is used to signal exclusively “cell 2” expressing the receptor for that ligand on its surface (see Figure 3), activating an easily detectable response—expression of GFP—in the second cell.



Figure 3. Circuit Design Modified for Quorum Sensing System Testing. The new circuit design splits the quorum sensing systems’ parts into 2 cells (one with the ligand and necessary export and modification machinery and one with the histidine kinase receptor and response regulator). Activation of ligand-creation in cell 1 via IPTG addition should result in fluorescence in cell 2. Note as well the resemblance between this system and that for insulin-mediated glucose regulation shown in Figure 1.

Additionally, one of the focuses of our project was on the implementation of combinatorial promoters to increase the functionality of the pResponse promoter. This component of the project originated from our original circuit design, in which a combinatorial promoter would have been necessary for correct functioning of the circuit (refer back to Figure 2), but we continued characterization of different combinatorial promoters during the summer, even after we had abandoned the original circuit design, in hopes that it could add functionality to our final circuit even though it was no longer necessary.