Arizona_State
The Problem
Brief Summary
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.
MOTIVATION
Suiting Up for Battle
Promoters are a region of DNA that initiates transcription of a specific gene. In bacteria, promoters contain 2 short sequence elements about 10 and 35 nucleotides upstream from a transcription start site. For our project, we utilize inducible promoters to initiate the transcription of regulator proteins and inducible GFP signals. Inducible promoters are a power tool in genetic engineering because the expression of genes operably linked to them can be turned on or off at certain stages of development of an organism or in a particular tissue [1] This allows us to track and analyze data to find orthogonality between senders and receivers. This is done when senders produce acyl-homoserine signals (AHLs) and attach to a regulator promoter in the corresponding receiver system. This allows for DNA binding and transcription initiation. This in turn allows for proteins to be made which then bind to the inducible promoter to allow GFP to be turned on. If there is no transcription of our regulator gene, aka no AHL attachment to the promoter of the regulator protein, our system won’t turn on. This will allow our team to run various experiments and see if it is working. This particular production of HSLs is just one one type of quorum sensing system out there and the type of system that is utilized in this project. We can test our system of senders and receivers for orthogonality by setting up induction experiments with different sender signals. A positive result means the sender signal will only turn on the GFP signal of one regulator gene, meaning the GFP is expressed in the system.
During the design conception of this project, we noticed that last year’s 2016 ASU iGEM team’s nonfunctioning receivers were not constructed properly on the DNA level. Since Last year’s team only had one receiver, it was difficult to find orthogonal pairs of sender-receivers. For this reason,we decided to design and synthesize new receivers for orthogonality testing. First, we researched into designing new promoters for AHL quorum sensing systems. In doing so, we found that the some of the receivers in the system researched last year did not have a proper inducible promoter. For example, some receiver systems did not even include an inducible promoter within their system; or they used a wrong binding site within the inducible promoter. For this reason, designing new inducible promoters for receivers was a top priority this year for our team.
Our team came across a paper by Spencer R Scott and Jeff Hasty from UC San Diego. They designed new inducible promoters that lead to better expression and easier cloning in their specific AHL related QS systems [2]. Due to this, our iGEM team utilized their thought and design process into our systems. The goal of our project is to successfully incorporate inducible promoters into our system to have them respond to HSLs and induce expression of GFP in E.coli. In addition, we hope to find undiscovered orthogonality between our senders and receivers. Hybrid promoters Ptra* and Prpa* were created by replacing the lux-box in the commonly used PluxI promoter with the tra-box and the rpa-box, respectively [2]. Using this idea, we created new receivers for our system with tra, rpa, and las genes to test in our experiments. In addition to this, new receivers of Bja [4], Aub [3], and Rhl [5] were created for testing. This was done by having a promoter from Lux and combining it with the specific regulator gene binding domain [2]. In addition to the inducible promoter, we also rearranged to order of our two part receiver system. The regulator and GFP were originally in that respective order. However, in our new receivers that orientation is switched. This was due to finding a leaky expression due to transcriptional read through of the receiver. By swapping the order, we can optimize the sequence to avoid transcriptional read through in our reporter gene.
Some broad descriptions of experiment ran are testing concentration of N-acyl homoserine lactone, testing combination of different senders, and testing induction and diffusion rates with senders and receivers.
Lost in Translation
Battle of the AHLs
AHL Disposal and Human Practices
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 | 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
F2620 Inductions
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:
- EsaI-BBa_K1670004
- LasI-Bba_C0178
- LuxI-BBa_C0161
- RhlI-BBa_C0170
- RpaI-BBa_K1421006
We added the induction data that we collected for these systems to their respective parts pages, which provides information on their compatibility with F2620.
REFERENCES
- Davis, René Michele, Ryan Yue Muller, and Karmella Ann Haynes. "Can the Natural Diversity of Quorum-Sensing Advance Synthetic Biology?" Frontiers in Bioengineering and Biotechnology 30th ser. 3 (2015)
- Borchardt, S.A., Allain, E.J., Michels, J.J. “Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials” Applied and Environmental Microbiology. 67.7. (2001); 3174-3179.
- Canton, Barry, Anna Labno, and Drew Endy. "Refinement and Standardization of Synthetic Biological Parts and Devices." Nature Biotechnology 26.7 (2008): 787-93.
- Scott, Spencer R., and Jeff Hasty. "Quorum Sensing Communication Modules for Microbial Consortia." ACS Synthetic Biology 5.9 (2016): 969-77.
- Miller, Melissa B., and Bassler, Bonnie L. “Quorum Sensing in Bacteria.” Microbiology. 55. (2001); 165-99
- Tait, Karen, Zoe Hutchison, Fabiano L. Thompson, and Colin B. Munn. "Quorum Sensing Signal Production and Inhibition by Coral-associated Vibrios." Environmental Microbiology Reports 2.1 (2010): 145-50.
- Grant, Paul, Dalchau, Neil, Brown, James R. “Orthogonal intercellular signaling for programmed spatial behavior.” Molecular Systems Biology 12.1 (2016):849.
- Steindler, Laura, Bertani, Iris, De Sordi, Luisa. "LasI/R and RhlI/R Quorum Sensing in a Strain of Pseudomonas aeruginosa Beneficial to Plants." Applied and Environmental Microbiology. 75.15: 5131–5140.
- Lindemann A. "Isovaleryl-homoserine lactone, an unusual branched-chain quorum-sensing signal from the soybean symbiont Bradyrhizobium japonicum." PubMed. 108.40 (2011):16765-70.
- Ahlgren NA. "Aryl-homoserine lactone quorum sensing in stem-nodulating photosynthetic bradyrhizobia." PubMed. 108.17 (2011):7183-8..
- Hoang, Hanh H. "The LuxR Homolog ExpR, in Combination with the Sin Quorum Sensing System, Plays a Central Role in Sinorhizobium meliloti Gene Expression." Journal of Bacteriology. 186.16 (2004): 5460-5472.
- Marketon, Melanie M. "Characterization of the Sinorhizobium meliloti sinR/sinI Locus and the Production of Novel N-Acyl Homoserine Lactones." Journal of Bacteriology. 184.20 (2002): 5686-5695.
- Case, Rebecca J., Labbate, Maurizio, Kjelleberg, Staffan. "AHL-driven quorum-sensing circuits: their frequency and function among the Proteobacteria." The ISME Journal. 2 (2008): 345–349.
- Winstanley, Craig, Fothergill, Joanne L. The role of quorum sensing in chronic cystic fibrosis Pseudomonas aeruginosa infections." FEMS Microbiology Letters. (2009) 1-9.
- Watson, William T. "Structural Basis and Specificity of Acyl-Homoserine Lactone Signal Production in Bacterial Quorum Sensing." Molecular Cell. 9.1 (2002) 685–694.
