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Jamboree/Project Abstract/Team Abstracts
From 2010.igem.org
Team Aberdeen_Scotland: The AyeSwitch: a translationally regulated genetic toggle switch in yeast
A novel genetic toggle switch regulated at the translational level was engineered in yeast that allowed the mutually exclusive expression of either green or cyan fluorescent protein. Using cell cytometry (FACS) and fluorimetry, we demonstrated in yeast the successful expression and translational regulation of a fusion of mRNA binding protein and fluorescent protein. These results, along with published parameter values, were used to predict via deterministic and stochastic models that the probability of successful bistability for our switch was 0.96%, but this could be improved theoretically to a maximum of 51.27% by limiting the range of variation of the most sensitive parameters. The models also predicted that co-operative binding of the mRNA binding protein to its mRNA stem loop was essential for generating switch-like behaviour. These results suggest that a translationally regulated genetic toggle switch is a viable and novel engineering concept applicable to medicinal, environmental and technological problems.
Team Alberta: GENOMIKON: An Educational Tool Kit for Rapid Genetic Construction
Building DNA is too hard. Democratizing Synthetic Biology will demand fundamental advances to make DNA construction easier and cheaper, thereby enabling broader access to biotechnology by the public. Our team has tackled this challenge with the design of an inexpensive self-contained kit called GENOMIKON, currently targeted for the high school and DIY communities but with clear potential for professional use. The kit contains prefabricated parts that are sequentially assembled on a solid support using cycle times of 5 min./part with a coupling efficiency of ~95%. The parts exist with sufficient diversity and quantity for hundreds of unique experiments. The kit is accompanied by an online resource that serves as lab manual, notebook, information management system and social network for the exchange of ideas. While similar in concept to our last year’s project, GENOMIKON differs in most technical aspects and is far superior in performance.
Team ArtScienceBangalore: Synthetic and Post-Natural Ecologies
In our second year as artists and designers at IGEM, we have decided to investigate the consequences of creating a Synthetic Ecology: an ecosystem in which organisms designed for a techno-scientific environment interact with organisms in the wild. C.elegans live on a diet of a variety of bacteria, E.coli being such strain. Genetically-modified E.coli can be fed to C.elegans which can then express any double stranded RNA of interest. The dsRNA can knock off specific genes in C.elegans. In our experiments, we are using C.Elegans as a marker to expres a range of external factors in two sets, temperature and IPTG. On a utilitarian level, our project investigates the use of C.elegans as a visual marker for changes in environmental conditions. On a more critical level, C.elegans is used to study the consequences of interactions between engineered organisms and the 'natural' world.
Team Baltimore_US: DIY-GEM: a path towards low cost high throughput gene synthesis
Synthetic biology research requires more cost effective approaches toward reagents and hardware accessibility. We are developing low-cost alternatives to existing hardware and enzymes in an attempt to expand participation in biological research and development. Our project expands the accessibility of Taq Polymerase by engineering it in a form compatible with BioBrick assembly. This allows use of the over-expressed enzyme from a crude bacterial extract in a PCR reaction at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel electrophoresis apparatus and a PCR thermal cycler. Enabling researchers to synthesize their own enzymes and having access to inexpensive tools will allow for increased participation among the DIY-bio community, stretch increasingly scarce educational funds, and allow rapid scale up of large scale gene synthesis projects.
Team BCCS-Bristol: agrEcoli: Smarter farming through bacterial soil fertility sensors
Fertiliser production is a major contributor to global carbon emissions, and excess fertiliser can cause immense damage to local ecosystems. Our lab has developed and characterised a cheap, versatile soil fertility sensor based on an E.coli chassis. It expresses fluorescent signals upon nutrient detection, producing a high-resolution nutrient distribution map of arable land. The ratio of two fluorescent signals allows farmers to quantify soil nutrient content. agrEcoli bacteria, encapsulated within a gel container to improve visibility and prevent escape, have been shown to work on soil in lab conditions. We have explored the marketing of our device, considering public perceptions of synthetic biology. BSim, our prize-winning modelling framework, has been extended to analyse our new biobricks’ behaviour within gel capsules. In addition, a new interface for BSim has improved its accessibility to the wider synthetic biology community, facilitating collaboration. agrEcoli optimises fertiliser use, saving farmers' money and reducing environmental damage.
Team Berkeley: Choachoa's Delivery Service
Single-celled phagocytic eukaryotes like Choanoflagellates are of great interest to developmental biologists because they may be the last living immediate precursor on the evolutionary tree to animals. These easy to culture and robust organisms are also a desirable eukaryotic chassis for synthetic biology, but there are few tools for delivering biomolecules into these organisms. So, we engineered E. coli to deliver proteins and/or DNA payloads into these bacteria-devouring eukaryotes. Once ingested, our E. coli are programmed to self-lyse and porate the phagosome, releasing their payloads into the cytosol. This delivery mechanism has the potential to deliver payload to any phagocytic organism with a cholesterol-based membrane. As part of our parallel software effort to rework the Clotho plugin environment and API, we made automatic biosafety handling an intrinsic feature of the core. Together, these tools provide a foundation for metazoan synthetic biology and a framework for improving safety in our field.
Team Bielefeld-Germany: MARSS - Modulated Acetosyringone Receptor Sensor System Defining Spiciness since 2010
The iGEM-Team Bielefeld is going to modulate an Agrobacteria receptor in Escherichia coli in order to detect capsaicin which is responsible for the hot taste of chilies. The intention is to make the spiciness in fare visible using a gradient light signal. The original receptor is the acetosyringone detection system of Agrobacterium tumefaciens. By using directed evolution, we aim to modulate the receptor binding domain to enable the interaction with similar phenolic substances like capsaicin. Brought into E. coli, this modulated system will induce light effects of different intensities - depending on the concentration of capsaicin respectively the spiciness of the sample. The capsaicin detection is a proof of principle concept. We aim to establish a system, which is characterized by a high sensitivity and specifity and is capable to replace slow and high priced diagnostics or analytic methods. The targets of the system could be allergy-triggers, explosives and toxins.
Team BIOTEC_Dresden: SensorBricks
SensorBricks is a reliable and modular system for antigen recognition, signal amplification and quantification. Initial steps of SensorBricks will focus on the detection of CD33 and other leukemic markers to increase diagnostic stringency. There are three major components in SensorBricks: (i) monoclonal antibodies that bind to an antigen of interest, (ii) a LuxI-Protein A fusion construct which non-specifically binds antibodies and produces the autoinducer N- Acyl homoserine lactone (AHL), and (iii) a Escherichia coli based biosensor which strongly amplifies the production of a fluorescence protein in the presence of AHL. By coupling signal detection to a genetic circuit, we would be able to amplify the signal in a quantifiable manner, allowing the identification of cancer markers expressed in minute quantities.
Team British_Columbia: A Multi-pronged Approach to Eliminating Staphylococcus aureus Biofilms Using Recombinant Bacteriophage and Biofilm-Degrading Enzymes
Biofilms are ubiquitous microbial communities that often display greater resistance and pathogenicity compared to individual microbes. Biofilms commonly cause complications in both industrial and medical settings and represent a significant source of morbidity and mortality. A synthetic biology approach to tackling biofilms has only recently been applied to Escherichia coli biofilms. To eliminate the more clinically relevant Staphylococcus aureus biofilms, our team aims to break new ground at iGEM by using S. aureus as a model host and developing a standard for genetically engineering bacteriophages. Our design incorporates DspB, a biofilm matrix-degrading enzyme into the Փ13 phage genome, which is altered to operate under the regulation of the S. aureus agr quorum sensing pathway and thus upon contact with biofilms. As a complement, we have also developed a mathematical model that simulates the dynamics of our system under different conditions.
Team Brown: Light Pattern Control of Cell Circuits
Biological manufacturing of complex compounds often requires the synthesis of many intermediate products. Production of these intermediates is currently triggered by inefficient methods, such as chemical inputs (tetracycline, estrogen-analogs, arabinose, etc) or drastic changes to the cellular environment (pH, oxygen levels, temperature, etc). On an industrial scale, this chemical induction requires large quantities of reagents and extensive purification, while environmental induction requires conditions that can adversely affect cell vitality and yield. To this end, we have designed an E. coli genetic circuit that can pass through four stable states of protein production triggered solely by ON/OFF patterns of light. To efficiently test the components of our circuit, we have also created a system for the transient delivery of transcription factors through the cell and nuclear membranes. With this production method, we can link multiple synthesis steps to a single, clean and rapidly scalable input.
Team Calgary: Translating Stress Into Success
The majority of projects in synthetic biology involve the over expression of recombinant proteins in microorganisms. A major stumbling block however, is often an inability to express functional protein. This situation is difficult to manage and troubleshoot as it is often unclear why expression is failing. We have designed a system that can accurately and visually report whether a gene is being transcribed and/or translated. The system also differentiates whether expression is failing due to misfolding in the periplasm or cytoplasm. In the case of misfolding, our system can also fine tune expression levels of a given protein to optimize production, increasing the likelihood of obtaining functional protein. To further understand protein misfolding we have built an equation-based, multivariant model of inclusion body formation. Finally, we used a series of podcasts to explore the social implications of our project in the context of the growing synthetic biology industry.
Team Caltech: Towards the Production of a Bioplastic Bioprinter and Design for a General Printing Framework
Our goal for the was to create and print a bioplastic, polyhydroxybutyrate (PHB), from soybean oil using E. coli. Our proposed design uses a radical crosslinking reagent to crosslink PHB monomers in cell lysate, released upon a light-induced lysis gene network. We hope to apply this printing ability to three-dimensional printing, offering a cheap alternative to current rapid-prototyping technologies. Our work involves characterizing an infrared promoter for light-lysis, experimenting with PHB production in cells, and the design of a dual-wavelength printing system. We discuss how this system could be generalized to create a framework for actuating groups of cells in any 3D volume to theoretically modulate behavior more complex than lysis. We also plan to apply special consideration to the ramifications of possible commercial enterprises developed in iGEM competitions with open source biological materials, such as BioBricks™.
Team Cambridge: E.glowli: a bioluminescent future
Bioluminescence is one of the most striking spectacles in the natural world. Taking genes from fireflies and Vibrio fischeri, the Cambridge team have constructed BioBricks which allow light output at a wide range of wavelengths. Firefly luciferase is already used as a reporter, but requires continual addition of the expensive substrate luciferin. We have created codon-optimised operons combining luciferase with a luciferin regenerating enzyme (LRE). This allows recycling of luciferin for sustained light output. In addition, we have submitted the first lux operon to the registry, taking genes from bacteria which form symbiotic relationships with squid. This is the first BioBrick to emit light without addition of substrate and can be used as a reporter with any promoter. These two approaches will allow cheaper assays with brighter signals. We also hope they will lay the foundations for natural light sources that help to address the energy crisis facing our planet.
Team CBNU-Korea: Design and Construction of Synthetic Minimal Chromosomes
Most of all bacteria have single circular chromosome. But some bacteria have two or more circular chromosomes. In Vibrio cholerae, there are two circular chromosomes, chromosome I and chromosome II, and each perfectly works as a chromosome. We’ve been motivated by V.cholerae’s two chromosome system. So we employed some essential genes, parA, parB, parS, dif, and origin of chromosome II and contstructed a tiny miniature of V.cholerae’s two chromosome system in E.coli, using BioBrick assembly method. Also, we built software and database of essential genes for designing of minimal synthetic chromosome and genome. Essential gene informations were gathered from some databases, DEG, EGGS, NCBI and java language was used. Our final goal is making useful, safe and stable synthetic minimal genome for Synthetic Minimal Cell. Although our project is feeble, we extremely believe that our project in this year will be worth first step for that.
Team Chiba: Eliminating the False-Input ~Genetic Double-Click System~
We daily double-click the icons to open the files or to exert the program: this is clearly distinguished from the single click, which is often for selecting or highlighting the program. This year, iGEM CHIBA is constructing genetic double-click system whose output is released only when the input (inducing agent) is given twice within a limited time. To discriminate double-click from two separated single-clicks, the 1st input is to be memorized temporarily. If the 2nd input is added before the memory gets lost, output will be produced. If the 2nd input is not added within the given time, the system will be reset to the original state. This mechanism could work as a sort of safety device; by requiring the 2nd ‘confirmation’ input, one can drastically reduce, or even eliminate, the frequency of false-inputs. This system could be useful in operating the potent or potentially-hazardous biochemical processes.
Team Cornell: OMG OMVs!
Outer membrane vesicles (OMVs) are natural secretions by gram-negative bacteria that can transport various proteins, lipids, and nucleic acids in interactions with mammalian host cells. OMV technology presents an affordable, non-toxic, and direct method of drug delivery and antigen tracking. We have designed a method for visualizing the interactions of mammalian cells with outer membrane vesicles by utilizing the ClyA surface protein as an attachment site for fluorescent proteins. The current goal of this project is to characterize the distribution of varying ClyA-fluorescent protein complexes on OMVs. Future work will be to develop a tracking system employing a ClyA-fluorescent protein construct for in vitro microscope imaging. An antibody fragment will also be attached to another ClyA complex, allowing the OMV tracking system to target specific regions of an organism. This method allows in vitro characterization of OMVs and provides integral data for developing a future OMV delivery platform in vivo.
Team Davidson-MissouriW: Foundational Advances in Biology and the Knapsack Problem
We focused on the Knapsack Problem which asks, "Given a set of weighted items and a knapsack of fixed capacity, is there some subset of these items that fills the knapsack?" Weighted items are represented by TetA alleles that confer measurably distinct levels of tetracycline resistance in E. coli. Excess TetA kills the cells; insufficient TetA can be screened by plating on tetracycline plates. Each TetA allele is coupled with a distinctive fluorescent gene, and both are flanked by variant lox sites. Cre protein can invert or excise floxed DNA, yielding different combinations of expressed TetA alleles. We constructed different TetA alleles by altering codon optimization and characterized the consequence of changing the order of two genes (TetA and RFP). Furthermore, we designed and tested a total of 11 new lox sites for site specific recombination. We developed several open access software tools for the wider synthetic biology community.
Team Debrecen-Hungary: The lipid sensor eukariotic toolkit
Eukaryotic synthetic biology has huge potential, yet it is still in need of more diverse molecular tools for defined gene regulation. Nuclear receptors are a conserved family of proteins responsible for sensing lipids; they may be viewed as lipid activated transcription factors. We have successfully developed a kit with a variety of lipid responsive domains (from H.sapines, D.melanogaster and C.elegans) for the rational construction of synthetic transcription factors. The domains respond only to predefined lipids and selectively activate predetermined gene expression. To characterize theses domains, we used standardized protocols for comparable measurements. In vivo gene expression was measured as a function of ligand concentration using luciferase activity. The potential for these tools is immense; e.g. from the ultra sensitive detection of lipid contaminants in the environment to the opportunity of titration specific gene expression canges in patients undergoing gene therapy.
