Difference between revisions of "Team:Arizona State/Description"

The Problem

• There are only around 4 widely-used N-acyl homoserine lactone (AHL) quorum sensing systems, while there are over 100 different systems found in nature
• AHL molecules are not completely system-specific, as crosstalk can occur between systems-this causes problems with developing functional genetic circuits
• AHL quorum sensing is used to regulate factors such as virulence and biofilm formation, potentially activating pathogenic bacteria

Brief Summary

Quorum sensing is the ability of bacteria to scan their surroundings and detect concentrations of their own population. A species of bacteria will produce an inducer protein, which generates chemical signals in the form of N-acyl homo-serine lactones (AHLs). When the AHL reaches a high concentration, the bacterial cells will respond by collectively activating a set of genes. In nature, these AHLs are able to drastically influence the growth behavior of bacterial cells, activating biofilm formation, bioluminescence, virulence, motility, etc.

The objective of our project is to design and test a variety of quorum sensing networks. We have developed a flexible testing platform in which the QS system is separated into two components designated the “Sender” and the “Receiver”. The AHL synthase is expressed in the Sender cell, while the inducible promoter and regulator are carried by a Receiver cell. When the Sender produces a signal, the HSL, it diffuses across cell membranes and activates the Receiver. In our current system, Receivers will express green fluorescent protein (GFP) in response to induction by Senders from different bacterial species. Ideally, the designed systems would have low amounts of interference and form a functional genetic circuit. Our team has built 10 senders and their respective receivers.

Our iGEM team is investigating the diverse applications that fit with our quorum sensing project. Some of the sub-projects include: investigating the Aub strain, which originates from unidentified soil bacterium; writing a white paper on addressing potential safety concerns with AHLs, a more comprehensive characterization of AHLs produced by our Senders using mass spectrometry, a direct comparison induction test on Senders using the well-characterized part F2620, and develop comprehensive safety procedures for the handling of AHLs. The white paper and safe disposal protocol are several of our human practices projects that the ASU team investigated during the course of the project.

MOTIVATION

N-acyl homoserine lactone (AHL) quorum sensing (QS) is a type of communication that allows bacteria to monitor their population density for the purpose of controlling various group activities such as virulence or luminescence. Synthetic biologists have taken advantage of the simplicity of the QS system to incorporate signal-processing pathways into genetic circuits. This project aims to determine pathways with minimal overlap (“crosstalk”) and engineer QS modules as a flexible tool for building layered genetic circuits. Crosstalk occurs when when a single regulator is activated by multiple varieties of HSL molecules, impeding successful operation of complex genetic circuitry that uses multiple quorum sensing pathways. Homologous AHL networks have been identified in over 100 species of bacteria, but only four have been used in synthetic systems reported to date. Our goal is to expand the QS toolbox and enable the implementation of higher-order, complex genetic circuitry in synthetic biology.

In addition to expanding the QS toolbox, our team felt that an in-depth investigation on the safety of AHLs would be valuable to future quorum sensing research. From the interviews and questionnaires that we conducted to industry experts, it was clear that AHLs were not a focal point in their safety reviews. However, from literature reviews, we found that there are many opportunistic pathogens that utilize AHL to activate virulence. A brief list is shown below:

Because there are potential pathogens that could become activated, proper safety measures in the development and production of AHLs were necessary. In our Human Practices, we gathered information about the dangers of AHLs from experts and literature, developed a safe disposal plan, and wrote a report on suggestions for the safety of AHLs. In addition, we added a safety sheet to our parts pages that we submitted to the registry.