Project Overview

Our project was split into 3 subprojects: AgrBDCA signal reception & Combinatorial Promoters, FsrABC/lamBCDA signal reception, and Ligand export systems.

AgrBDCA Signal Reception & Combinatorial Promoters

AgrBDCA is a quorum sensing system found to regulate virulence factors in Staphylococcus aureus. In this system, agrC and agrA function as a histidine kinase receptor and a response regulator respectively. This subproject aimed to introduce agrC and agrA in functioning form into E. coli. The circuit used for this subproject is depicted below.

Figure 1 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.

Additionally, this subgroup focused on prototyping various combinatorial promoters--those which accept multiple inputs in order to determine an output state. This part of the subproject stemmed from the original overall project idea, which was to characterize a negative feedback loop with extracellular components and required a combinatorial promoter to control production of the ligand exported out of the cell. Since work had already begun on that project, it was continued, even after the shift in project design and focus, in hopes that it will later be incorporated again in the circuit.

LamBCDA & FsrABC Signal Reception

lamBCDA is another quorum sensing system native to Lactobacillus plantarum that shares homology with the agrBCDA quorum-sensing system in S. aureus. Normally, it regulates adherence to surfaces and cell morphology. The components mirror those of agrBCDA, shown in Figure 1, exactly. (i.e. lamA is the response regulator, lamC is the histidine kinase receptor, etc.)

FsrABC is a third quorum sensing system homologous to the agrBCDA system of S. aureus and is also believed to regulate virulence factors involved in biofilm formation in Entereococcus faecalis. In this system, FsrC and FsrA function as histidine kinase receptor and response regulator respectively. FsrB is a membrane protein that is processed to form a cyclic 11-amino-acid polypeptide (see Ligand Export Systems).

Figure 2 The fsr quorum sensing system also has 2 components involved in signal reception: fsrC as the histidine kinase receptor and agrA as the response regulator. However, unlike the agr and lam systems, which have separate domains encoding a short peptide ligand-precursor and a membrane-protein to modify said ligand-precursor, the fsr system encodes a single membrane protein (fsrB), which is eventually cleaved to form the peptide ligand.

Ligand Export Systems

While the previous 2 subgroups focused primarily on the signal reception aspects of their respective quorum sensing systems, this subgroup focused on the signal transmission aspects of those quorum sensing systems. Specifically, we focused on importing into E. coli the genes for the peptide ligand and any "accessory" proteins (e.g. agrB, lamB) necessary for export of and modification of that peptide into its functional ligand form.

Specifically, within the AgrBDCA system, it is believed that agrB is a membrane protein that exports and modifies a short polypeptide sequence (agrD) into a polypeptide ligand to be bound by agrC, the histidine kinase receptor. Similarly, the lamBDCA system is thought to function in a very similar way. The fsrABC's export system however, is believed to function by cleaving off a small fragment of fsrB, which is localized in the cell membrane, to become the peptide ligand (see Figure 2 above).