Team:Caltech/Project/Details
From 2014.igem.org
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Project Overview
Project Details Materials and Methods The Experiments Results Data Analysis 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 [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 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). [3] 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 4). ![]()
agrBCDAThe agrBCDA quorum sensing system is 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. Figure 1 contains shows the reception mechanism for this system.![]()
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. lamBCDAThe lamBCDA system is a quorum sensing system native to Lactobacillus plantarum 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?].fsrABCThe fsrABC system is a quorum sensing system native to Enterococcus faecalis 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. |