Team:Caltech/Project

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, 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.

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 1). 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 1. 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 2), activating an easily detectable response—expression of GFP—in the second cell.

Figure 2. 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.

Quorum Sensing Systems Investigated

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 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 3. 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.

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]

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 4).

Figure 4 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. [4]