Difference between revisions of "Team:Arizona State/Description"
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Quorum sensing (QS) is at the foundation of a wide range of high-impact bioengineering efforts such as creating new biosensors and health monitoring devices while providing a toolbox of reusable genetic components that can be plugged into circuits at will (Kwok, 2010). QS involves systems of bacteria that use sender and receiver molecules to communicate gene expression when the bacteria reach optimal densities. In doing so, quorum sensing allows bacteria to express specific genes at a high population density that results in beneficial phenotype expression.There are many different types of QS sensing molecules and in this research we will be working with a type of chemical signal called acyl-homoserine lactones (HSLs). When these signals are received by the surrounding bacteria, the signal is transduced. Creating genetic circuits is done by characterizing genetic sequences that perform needed functions and combining them into devices that are inserted into cells (Kwok, 2010).
A major problem in engineered systems is crosstalk. This is when different species of senders can activate a noncanonical receiver's promoter. When QS pathways operate without communication between unwanted cells, the pathways are orthogonal and potentially viable options for bioengineering new synthetic circuits. A sender is defined as cell that expresses AHL synthase, while a receiver is a cell that includes an inducible promoter that initiates transcription of a gene and regulator controls the expression of one or more genes. Researchers want to find systems that are completely orthogonal, in efforts to enhance efficacy while maintaining specificity [7]. The application of defining cross-talk might lead to more sophisticated intracellular communication [7]. In addition, these circuits may then be engineered to detect specific combinations of input signals that could be used to engineer multi-strain, self monitoring microbial populations that perform energetically costly metabolic processes in a single culture (Davis et al., 2015).
The objective of our project is to design and test a variety of quorum sensing networks. This includes creating new receivers for our system, as well as researching various concentrations of AHL signals. 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 3 new receivers.
Our iGEM team is investigating the diverse applications that fit with our quorum sensing quest. Some of the side quests include: new receivers with hybrid promoters, concentration of N-acyl homoserine lactone (AHLs), combinations of different senders, induction and diffusion rates, mathematical models and the case of the disappearing mCherry.
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 | 3D-Model |
|---|---|---|---|
| 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.
AHLs share the same basic structure, with a lactone ring, an N-acyl and ketone group. The defining R group lies in the acyl tail, which is the primary determinant in its transcription factor binding affinity. The graphic below demonstrates the categorization of the AHLs produced by the 10 studied systems
The ASU team would like this experiment to be considered for the Gold Medal Requirement of improving the characterization of a previously existing BioBrick part. All 10 systems were studied in an induction test. The part BBa_F2620 (designed by Barry Canton from MIT) was used to induce production in the Lux AHL system and test induction in any other AHL systems. By studying interactions between the 10 constructed Senders and F2620, we were able to analyze the systems for potential orthogonality. The resulting part collection allows direct comparison in AHL induction between multiple systems. The Part Numbers for these Senders are BBa_K2033000, BBa_K2033002, BBa_K2033004, BBa_K2033006, BBa_K2033008, which correspond to the Aub, Bja, Bra, Cer, and Sin systems. We believe that this Part Collection is a valuable addition to the registry, because while small in size, it contributes 5 Senders to the registry which contains around 6 Senders. In addition, this will add information about these new systems to a well-characterized and widely-used part in F2620. Not only will this determine interactions between systems, but also, provide characterization information for any future quorum sensing research involving those systems.
We also created Senders for the Esa, Las, Lux, Rhl, and Rpa systems, which already exist in the registry. These correspond to the following parts:
We added the induction data that we collected for these systems to their respective parts pages, which provides information on their compatibility with F2620.
