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Brief Description

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. Currently, our team has built 10 senders and 7 receivers, several of which have been shown to be functional in E. coli. Testing for the remaining systems is still underway, and more receivers are still being cloned.

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, to decipher its organism of origin; making a “super quorum sensing” E. coli that is engineered to respond to wider variety of AHLs; a more comprehensive characterization of AHLs produced by our Senders using mass spec; and develop comprehensive safety procedures for the handling of AHLs. The latter is one of our several human practices projects that the ASU team investigated during the course of the project.

N-Acyl Homoserine Lactones

AHL quorum sensing has a myriad of different systems. A total of 10 systems were investigated in this project

AHL System	Bacteria of Origin	AHL Name
Aub	Unknown	N-(2-oxooxolan-3-yl)dodecanamide
Bja	Bradyrhizobium japonicum	3-methyl-N-[(3S)-2-oxooxolan-3-yl]butanamide
Bra	Paraburkholderia kururiensis	(3S)-3-[(2-oxo-3-phenylpropyl)amino]oxolan-2-one
Cer	Rhodobacter sphaeroides	(Z)-3-hydroxy-N-[(3S)-2-oxooxolan-3-yl]tetradec-7-enamide
Esa	Erwinia stewartii	3-oxo-N-[(3S)-2-oxooxolan-3-yl]hexanamide
Las	Pseudomonas aeruginosa	3-oxo-N-(2-oxooxolan-3-yl)dodecanamide
Lux	Vibrio fischeri	3-oxo-N-(2-oxooxolan-3-yl)hexanamide
Rhl	Rhizobium leguminosarum	N-(2-oxooxolan-3-yl)butanamide
Rpa	Rhodopseudomonas palustris	(S)-(−)α-amino-γ-butyrolactone
Sin	Sinorhizobium meliloti	N-[(3S)-2-oxooxolan-3-yl]octanamide*

*Sin system produces 6 different variants of AHL. The 3D structures of all the Sin compounds can be found here.

All systems were investigated in an inductions investigation. The part BBa_F2620 was used to induce production in the Lux AHL system and test induction in any other AHL systems. Should induction occur, then possible interference between systems are conceivable, which may have implications towards any use of that system. The resulting part collection allows direct comparison in AHL induction between multiple systems.

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.

GOLD MEDAL WORK

To be added

REFERENCES