Team:Caltech/Project

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Revision as of 21:04, 17 October 2014



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Project Description
Project Overview

Project Details

Materials and Methods

The Experiments

Results

Conclusions

References

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 (Figure 1), 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.



Figure 1. Proper Glucose Regulation. In a normal human, insulin is produced in the beta cells of the pancreas, post-translationally modified, and then shipped out of the cell via exocytosis. It is then picked up by tyrosine kinase receptors on muscle cells, activating a signal transduction pathway ultimately leading to glucose uptake from the blood via endocytosis. In a patient with diabetes, depending on the type of diabetes, some aspect of this pathway stops functioning.

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 2). 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 2. 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 3), activating an easily detectable response—expression of GFP—in the second cell.



Figure 3. 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. Note as well the resemblance between this system and that for insulin-mediated glucose regulation shown in Figure 1.

Additionally, one of the focuses of our project was on the implementation of combinatorial promoters to increase the functionality of the pResponse promoter. This component of the project originated from our original circuit design, in which a combinatorial promoter would have been necessary for correct functioning of the circuit (refer back to Figure 1), but we continued characterization of different combinatorial promoters during the summer, even after we had abandoned the original circuit design, in hopes that it could add functionality to our final circuit even though it was no longer necessary.