Team:Caltech/Project/Details

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<a id='modeling' href="https://2014.igem.org/Team:Caltech/Modeling"style="color:#000000"> Modeling</a></td>
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<a id='modeling' href="https://2014.igem.org/Team:Caltech/TXTL"style="color:#000000"> TXTL Promoter Characterization</a></td>
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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>
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<a href = "https://2014.igem.org/Team:Caltech/Project/Results">Our Results</a>
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<a href = "https://2014.igem.org/Team:Caltech/Project/Data">Data Analysis</a>
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<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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<h3>Quorum Sensing Systems Investigated</h3>
<h3>Quorum Sensing Systems Investigated</h3>
<i>agrBDCA system</i>
<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]. <b>Figure 3</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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<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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<img src="https://static.igem.org/mediawiki/2014/a/a2/Caltech_agrSys.png" width=80%>
<img src="https://static.igem.org/mediawiki/2014/a/a2/Caltech_agrSys.png" width=80%>
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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 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>
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<i>lamBDCA System</i>
<i>lamBDCA System</i>
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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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<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>
<i>fsrABC System</i>
<i>fsrABC System</i>
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<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 4</b>).</p>
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<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>
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<table width=70%><tr><td><b>Figure 4 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. [4]</td></tr></table>
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<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>
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Three quorum sensing systems were chosen to test in <i>E. coli</i>:
 
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<h3>agrBCDA</h3>
 
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The agrBCDA quorum sensing system is native to <i>Streptococcus aureus</i> 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. <b>Figure 1</b> contains shows the reception mechanism for this system.
 
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<img src="https://static.igem.org/mediawiki/2014/5/55/AgrABCD_circuit_diagram.jpg" width="350px">
 
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<table width=70%><tr><td><b>Figure 1</b> The agr quorum sensing system, like most quorum sensing systems, has 2 components involved in signal reception: a histidine kinase receptor (agrC) that detects the ligand outside the cell and a response regulator (agrA) that is phosphorylated inside the cell by the activated histidine kinase receptor.</td></tr></table>
 
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In the endogenous system, the P2 promoter is actually found before the entire operon, so that the entire system forms a self-inducing, positive feedback loop. In the context of the agrBCDA as a quorum sensing system regulating virulence factors, it makes perfect sense as a system that will "push itself forward" and progress, once the Streptococcus reach a critical density. However, in our system, we are controlling ligand production via a pTet promoter and using the P2 promoter only to activate expression of the reporter.
 
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<h3>lamBCDA</h3>
 
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The lamBCDA system is a quorum sensing system native to <i>Lactobacillus plantarum</i> that is homologous to the agrBCDA system mentioned above. lamB is purported to be a membrane protein assisting in the export and post-translational modification of lamD, the signaling peptide precursor, while lamC is the histidine kinase membrane receptor protein responsible for detecting presence of lamD and, upon doing so, phosphorylating lamA, the response regulator, which proceeds to activate transcription of any gene downstream of the pLam promoter [is that its name?].
 
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<h3>fsrABC</h3>
 
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The fsrABC system is a quorum sensing system native to <i>Enterococcus faecalis</i> 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, while the FsrB protein is a membrane protein that is cleaved to form GBAP, the peptide mature signaling ligand.
 
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Latest revision as of 21:42, 17 October 2014



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Materials and Methods

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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 Investigated

agrBDCA system

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]. 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.



Figure 1. agrBDCA Quorum Sensing System. 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.
lamBDCA System

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 System

The 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).



Figure 2. fsrABC quorum sensing system 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]