Team:Caltech/Project/Details
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<a id='home' href="https://2014.igem.org/Team:Caltech"style="color:#000000">Home </a> </td> | <a id='home' href="https://2014.igem.org/Team:Caltech"style="color:#000000">Home </a> </td> | ||
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<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; modeling.style.color='white'" onMouseOut="this.bgColor='white'; modeling.style.color='black'" bgColor=white> | <td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; modeling.style.color='white'" onMouseOut="this.bgColor='white'; modeling.style.color='black'" bgColor=white> | ||
- | <a id='modeling' href="https://2014.igem.org/Team:Caltech/ | + | <a id='modeling' href="https://2014.igem.org/Team:Caltech/TXTL"style="color:#000000"> TXTL Promoter Characterization</a></td> |
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; notebook.style.color='white'" onMouseOut="this.bgColor='white'; notebook.style.color='black'" bgColor=white> | <td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; notebook.style.color='white'" onMouseOut="this.bgColor='white'; notebook.style.color='black'" bgColor=white> | ||
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- | <a href = "https://2014.igem.org/Team:Caltech/Project"> | + | <a href = "https://2014.igem.org/Team:Caltech/Project">Project Overview</a> |
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- | <b>< | + | <b><font size=+1>Project Details</font></b> |
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<a href = "https://2014.igem.org/Team:Caltech/Project/Methods and Methods">Materials and Methods</a> | <a href = "https://2014.igem.org/Team:Caltech/Project/Methods and Methods">Materials and Methods</a> | ||
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<a href = "https://2014.igem.org/Team:Caltech/Project/Experiments">The Experiments</a> | <a href = "https://2014.igem.org/Team:Caltech/Project/Experiments">The Experiments</a> | ||
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- | <a href = "https://2014.igem.org/Team:Caltech/Project/Results">Results | + | <a href = "https://2014.igem.org/Team:Caltech/Project/Results">Our Results</a> |
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<br><br> | <br><br> | ||
<a href = "https://2014.igem.org/Team:Caltech/Project/Conclusions">Conclusions</a> | <a href = "https://2014.igem.org/Team:Caltech/Project/Conclusions">Conclusions</a> | ||
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- | <p> | + | To build our circuit to characterize these systems, three quorum sensing systems from three different species of gram-positive bacteria were chosen to test in <i>E. coli</i>: |
+ | <br><br> | ||
+ | <h3>Quorum Sensing Systems Investigated</h3> | ||
+ | <i>agrBDCA system</i> | ||
+ | <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 [1]. <b>Figure 1</b> 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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+ | <br> | ||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/a/a2/Caltech_agrSys.png" width=80%> | ||
+ | <br> | ||
+ | <table width=70%><tr><td><b>Figure 1. 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> | ||
+ | </center> | ||
+ | |||
+ | <i>lamBDCA System</i> | ||
+ | <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). [2]</p> | ||
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+ | <i>fsrABC System</i> | ||
+ | <p>The fsrABC quorum sensing system, native to <i>Enterococcus faecalis</i>, is a QS system believed to regulate virulence factors involved in biofilm formation in its native host. In this system, fsrC and fsrA proteins function as histidine kinase receptor and response regulator respectively, functioning similarly to their analogous components in the agrBDCA system (agrC and agrA).The primary difference between the agrBDCA and fsrABC systems lays in their ligand-production components. In the fsrABC system, the fsrB protein is a membrane protein that is cleaved and then biochemically modified to form GBAP, a cyclic 11-amino-acid polypeptide that is the peptide mature signaling ligand (see <b>Figure 2</b>).</p> | ||
+ | |||
+ | <br> | ||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/8/89/Caltech_fsrSys.png" width=60%> | ||
+ | <br> | ||
+ | <table width=70%><tr><td><b>Figure 2. fsrABC quorum sensing system</b> The fsr system differs from the agr & lam systems in that GBAP, its signaling peptide, is actually formed from a precursor peptide derived from cleaving the membrane protein fsrB. The exact biochemical pathway transforming the cleaved precursor peptide into the mature signaling peptide is yet unknown. GBAP proceeds to bind the histidine kinase receptor fsrC, phosphorylating FsrA, activating further transcription of the operon, similar to the other 2 quorum sensing systems. [3]</td></tr></table> | ||
+ | </center> | ||
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Latest revision as of 21:42, 17 October 2014
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Project Overview
Project Details Materials and Methods The Experiments Our Results Conclusions References |
To build our circuit to characterize these systems, three quorum sensing systems from three different species of gram-positive bacteria were chosen to test in E. coli:
Quorum Sensing Systems InvestigatedagrBDCA systemThe 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 [1]. Figure 1 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 S. aureus 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. ![]()
Additionally, we investigated the lamBDCA QS system, a quorum sensing system native to Lactobacillus plantarum. In its native environment, the lamBDCA system regulates adherence of L. plantarum to surfaces and cell morphology. Of note is that it shares significant homology with the agrBDCA quorum-sensing system in S. aureus: 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). [2] fsrABC SystemThe fsrABC quorum sensing system, native to Enterococcus faecalis, is a QS system believed to regulate virulence factors involved in biofilm formation in its native host. In this system, fsrC and fsrA proteins function as histidine kinase receptor and response regulator respectively, functioning similarly to their analogous components in the agrBDCA system (agrC and agrA).The primary difference between the agrBDCA and fsrABC systems lays in their ligand-production components. In the fsrABC system, the fsrB protein is a membrane protein that is cleaved and then biochemically modified to form GBAP, a cyclic 11-amino-acid polypeptide that is the peptide mature signaling ligand (see Figure 2). ![]()
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