Jamboree/Project Abstract/Team Abstracts

From 2010.igem.org

Revision as of 15:19, 21 September 2010

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.

Team DTU-Denmark: Bi[o]stable – Engineering a bistable switch

The aim of this project is to engineer a genetic bi-stable switch that produces two different, mutually exclusive outputs when given two different inputs. The switch is based on the repressor-anti-repressor system of the salmonella phages Gifsy1 and Gifsy2 and the λ-phage anti-termination system. The latest induced output will remain stable through generations, even once the input ceases, due to the phage regulatory systems. We present the framework for this development and characterize the regulatory mechanisms by using fluorescent proteins as the reporter (outputs). The dynamics of the system have been modeled and we have also attempted to characterize and submit the promoters, repressors and anti-repressors from the salmonella phages, as well as the two anti-terminator proteins from the lambda phage, as BioBricks. We have hereby demonstrated the engineering of a multipurpose bi-stable switch sensor/reporter tool that can have numerous applications.

Team Duke: Engineering a Robust Genetic Switch

Our project aims to produce a genetic transistor which, unlike most bistable switch mechanisms available to synthetic biologists, does not exhibit basal regulatory noise. The transistor will be based on a protein sequestration pathway that uses leucine zippers (bZIPs) Fos and Jun alongside synthetically designed dominant negatives thereof, eliciting a response dynamic similar to a signal titration. Furthermore, we intend to apply such transistors to function as signal amplifiers due to the ultrasensitive responses that can be generated in this mechanism. For the application of this project and others, we are also developing a high throughput gene expression screen for synthetic gene libraries and codon variants, allowing for the possibility of tunable gene expression levels.

Team ECUST-Shanghai: _

Team Edinburgh: Communicating Through Bridges: Bridging with Biology, Bridging with Light, Bridging with People

The engineering equivalent of Genetic Engineering is to get a bunch of concrete and steel, throw it into a river, and if you can walk across it, call it a bridge. Synthetic biology and iGEM have long?attempted to refine this process of 'bridge-building'. The 2010 University of Edinburgh team has applied this idea comprehensively throughout their project. The BRIDGE protocol (BioBrick Recombineering In Direct Genome Editing) is a protocol for markerless insertion of BioBricks onto the bacterial chromosome, which will bridge ideas and reality in synthetic biology. Bacterial BRIDGEs aim to foster non-chemical means of communication between bacteria by pairing light-producing and light-sensing BioBricks; future teams may make use of them in a variety of novel applications. Finally, human BRIDGEs examine synthetic biology as ways of thinking and the permeation of human aspects, bridging the so-called 'divides' between disciplines and individuals. The question is... how do you think?

Team EPF_Lausanne: Asaia, the pink force against malaria

Malaria is a tropical disease that kills more than 1 million people each year and no effective cure or vaccine exists yet. The EPFL iGEM project aims to stop malaria propagation by acting on the vector: the mosquito. We are engineering Asaia, a bacterium that naturally lives in the mosquito's gut, to express an immunotoxin that can prevent the malaria agent Plasmodium falciparum from infecting the mosquito, thereby eliminating the transmission of this parasite to humans. Asaia is an organism that is easy to grow and genetically manipulate. We are establishing Asaia as a new chassis so that future iGEM teams can quickly and efficiently engineer new and more potent Asaia strains. This will provide the synthetic biology community with a useful tool in the fight against malaria and other mosquito-borne diseases.

Team ESBS-Strasbourg: A light-controllable specific protein degradation system as new standard for synthetic biology

The aim of our project is to engineer a new fundamental component that could be universally used to build more complex or more controllable biological circuits inside chassis organisms. This new component consists of the E. coli protease ClpXP to which the phytochrome B (PhyB) of Arabidopsis thaliana is fused. Any given protein can be degraded as long as it is fused with the Phytochrome Interacting Factor (PIF)-degradation tag biobrick. The activity of this system is tightly controlled and switchable by light inducement.

Team ETHZ_Basel: E. lemming – a remote controlled living robot

We control the movement of a single E. coli cell by light. In wild type E. coli flagella movement is controlled by proteins of the chemotaxis pathway, so called Che proteins. In our engineered cells one of these Che proteins is fused to a synthetic light-sensitive localization system. Two external inputs – red light and far red light - induce the relocation of the fused proteins, thus reversibly changing flagella movement direction. Cells, imaged by bright field microscopy, are automatically detected and tracked while a closed loop controller guides the cell into a user defined direction by autonomously sending light inputs. This makes our engineered cell the smallest remote controllable living robot on earth.

Team Freiburg_Bioware: A Modular Virus Construction Kit for Therapeutic Applications

Gene delivery using viral vectors holds great promise for the treatment of acquired and inherited diseases. The human Adeno-Associated Virus (AAV) is a small, non-pathogenic, single-stranded DNA virus gaining increasing attention being both versatile and effective. Taking current knowledge into account, we generated a recombinant, modularized, BioBrick-compatible AAV ‘Virus Construction Kit’. We provide parts for modified capsid proteins, targeting modules, tumor-specific promoters, and prodrug-activating enzymes as well as readily assembled vectors for gene delivery and production of non-replicative virus particles. The viral tropism is altered by N-terminal fusion or by loop replacement of the capsid proteins. Functionality of viruses constructed from our kit was demonstrated by fluorescent protein expression in infected cells and by prodrug-induced killing of tumor cells upon viral delivery of a thymidine kinase. Incorporating multiple layers of safety, we provide a general tool to the growing field of personalized medicine and demonstrate its use in tumor therapy.

Team Freiburg_Software: SynBioWave 2.0 – A Collaborative Toolkit for Synthetic Biology

SynBioWave is an open-source, Synthetic Biology software suite based on Google’s open-source communication tool Wave. SynBioWave enables research collaboration by real-time sharing of parts, design and documentation. Moreover, biologists can record and share the process of creating research data. Last year our team developed the basic SynBioWave robot. This year we ported the main program (Robot) to Wave API 2.0 and improved user friendliness, separated the input and output from the sequence database operations by creating a linked wave for data storage. We also provide the “blueprint-robot”, a framework easing new robot development. Furthermore, we are adding new functionality by creating add-on robots that perform tasks such as BLAST-searches, ORF-finding, translation, sequence alignments and restriction site mapping. The main robot is available at SynBioWave@appspot.com, the source code at http://synbiowave.sourceforge.net and the homepage of the project is http://www.synbiowave.org.

Team Fudan-Shanghai: _

Team Gaston_Day_School: Construction of a Biological Iron Detector in a Secondary School Environment

Our team’s project was to create a biological iron detector using techniques and procedures available to an ordinary high school laboratory that replicate methods used in university research laboratories. We constructed our reporter by combining an iron-sensitive promoter with a red fluorescent protein (RFP) coding sequence. We chose RFP because of its high visibility and easy detection. Although the assembly was successful, the resulting detector is leaky with measurable RFP even in conditions with no iron present. In our lab environment, we found that it was necessary to work with relatively high concentrations of bacteria and DNA. We developed simplified procedures for transformations, digests, and ligations, but we continue to face problems with DNA visualization and measuring the pigments from the bacteria.

Team Georgia_State: Pichia pastoris: A Novel Chassis for iGEM

The methylotrophic yeast, Pichia pastoris, is increasingly used as an alternative host for heterologous protein production. P. Pastoris is advantageous because it is able to perform eukaryotic post-translational modifications, produce high yields of recombinant protein, and it is genetically similar to Saccharomyces cerevisiae. (Cereghino and Cregg, 2000). The 2010 Georgia State team believes P. pastoris would be an excellent chassis for the iGEM competition. The purpose of this project is to provide a tool box of parts necessary for the genetic manipulation of this organism. These parts include a variety of promoter systems, multiple selectivity options, and a plasmid backbone. In addition, the tool box will be used to produce a flu virus antigen in P. pastoris as a representation of the applicability of this system. These contributions will enable future users to maximize the use and further explore the incredible potential P. pastoris has to offer.

Team GeorgiaTech: Inducing a Thermogenic Response to Cold-shock in Bacteria

Alternative Oxidase (AOX) is a terminal oxidase protein found in the respiratory chain of various organisms ranging from aquatic prokaryotes to plants and animals. In the AOX pathway, electrons are transferred from ubiquonone to AOX, and then directly used to reduce oxygen. The drop in the electric potential energy of the electrons transferred from AOX to oxygen is dissipated as heat. Our project has focused on 1) cloning the AOX gene from a thermogenic plant (Sacred Lotus) into E. coli to induce a thermogenic response to a cold-shock, and 2) calculating a theoretical rate of heat production per bacterial colony to select for an appropriate calorimetric technique. Further, numerical methods in MATLAB will be employed to model the steady-state temperature profile of the synthetic bacterial colony, and to potentially corroborate later experimental findings. Engineering a controlled thermogenic response in bacteria could lead to improved bacterial functioning in cold shock environments.

Team Groningen: _

Team Harvard: iGarden: an Open Source Toolkit for Plant Engineering

The Harvard iGarden is a venture into plant engineering. We aim to create a toolkit for the cultivation of a personalized garden containing features introduced through synthetic biology. In addition to a "genetic fence" designed to prevent the spread of introduced genetic material, we have developed three independent features to be included in this toolkit - inclusion of novel flavors, knockdown of plant allergens, and modification of petal color. All parts are BioBrick compatible and introduced into plants through agrobacterium-mediated transformation, using existing plant vectors modified with the BioBrick multiple cloning site. The Harvard iGarden is an effort to raise public awareness of synthetic biology, production of food, and how the two can intertwine. We envision the iGarden as a medium through which the non-scientist can see the power and potential of synthetic biology, and apply it to everyday life.

Team Heidelberg: miBricks: DNA is not enough

The key to successful gene therapy is integration of tissue specificity and fine-tuned target gene expression. The iGEM Team Heidelberg 2010 unlocks the world of synthetic microRNAs, since focusing solely on DNA has often been inconvenient for medical purposes. We engineered a toolkit for standardized measurements of interactions between artificial miRNAs and their binding sites. From this data we were able to compute an in silico model integrating binding site properties and knockdown percentages. Thus, the expression level of any gene of choice could be arbitrarily adjusted by employing the corresponding binding site design. To produce tissue specific miRNA gene shuttles, we developed an evolution-based method for synthesis of new adeno associated viruses. This enabled us to overcome the natural limitations of virus selectivity. In the future, miBricks could be applied for treatment of diseases like Diabetes and Hemophilia, opening the doors to new Synthetic Biology based medical approaches.

Team HKU-Hong_Kong: The bio-safety net

Our team’s project is a “bio-safety net” that limits the survival of bacteria according to tailored conditions. Bacteria could be designed to perform promising tasks, such as the biodegradation of oil to clean up oil spills. Yet, there are risks associated with the possibility of living bacteria performing undesired activities. Our goal is to introduce a “bio-safety net” that will be applicable to virtually all genetically engineered bacteria as a vital termination step after their tasks have finished. We have made this possible by introducing a "suicide" mechanism, that will be triggered under specific conditions. By combining different promoters, the system can respond to changes in  environmental factors and control expression specific to chosen factors. Such mechanism can be easily assembled and incorporated to bacteria through the use of biobricks.

Team HKUST: Engineered Lactobacillus against S. aureus Infection

Our project aims at establishing an interspecies quorum quenching system in which engineered Lactobacillus can sense and reduce the virulence of potentially pathogenic Staphylococcus aureus. To accomplish this, we are constructing chimeric quorum sensing receptors that can localize on Lactobacillus membrane and detect autoinducing peptides (AIPs) released by S. aureus. The ligand binding to the chimeric receptor will trigger downstream plnABCD pathway and initiate the synthesis and secretion of RNAIII inhibiting peptide (RIP), a heptapeptide with proven effectiveness in attenuating S. aureus virulence. The possibility of achieving this lies in the structural homology of the catalytic domain of the quorum sensing receptors in Lactobacillus and S. aureus. Both receptors belong to the HPK10 subfamily of a two-component histidine kinase family. Attenuation of S. aureus virulence by quorum-sensing inhibitors should not yield a strong selective pressure for development of resistance, and would therefore be an attractive concept for preventive medicine.

Team HokkaidoU_Japan: Dr. E. coli: World Smallest Protein Injector

Our project is on Type lll Secretion Apparatus which is one of the most amazing biological devices. It can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. This apparatus which looks like a syringe is an organelle of pathogenic gram-negative bacterium such as Salmonella and Yersinia. We are aiming at making this device available for E. coli. Because it will not involve usage of pathogenic strains, it will be safer to use.  To transfer T3SS functionally from Salmonella to E.coli it is essential to integrate at least 40kb of DNA fragment coding more than 20 proteins. So we will make suggestions about how to optimize E.coli transformation method for large size DNA fragments. Also we will show how to construct protein for secretion and how to measure if it is really secreted using GFP.

Team Hong_Kong-CUHK: Bio-cryptography: information en/decryption and storage in E. cryptor

Data encryption and storage has always been an important branch of research in computer engineering. In our project, we explored the possibility of harnessing a biological system as an alternative solution for data en/decryption and storage. By using E. coli, we engineered and devised a prototype, dubbed E. cryptor, for 1) bio-encryption and -decryption with error checking; and 2) data storage in a bacterial system. In the age of synthetic biology, designed microorganisms may carry a specific DNA barcode to be distinguished from their natural counterparts. Our system could turn such barcode into more than simply a tag. In the future, can we also store text, pictures, and even videos into these tiny bacteria and protect the contents?

Team IBB-Pune: _

Team IIT_Delhi_1: Dr.coli

The use of bacteria for sensing applications has been around for a while now, and they have been used to produce recombinant proteins as needed for even longer time. The current project focuses on integrating these two components to create a device capable of responding to external stimuli in the form of quantitative protein production. For this device to function, it needed to be capable of producing and secreting the protein extracellularly. Further the dynamics of elicitor interaction with the bacteria in a flow stream and concomitant product release have also been a part of the study. We believe that such a system can play a major role in drug delivery systems that treat as needed and further in creating artificial glands for diseases such as insulin.

Team IIT_Madras: _

Team Imperial_College_London: Parasight – Parasite detection with a rapid response

More than two billion people around the world live with unrelenting illness due to parasites” - WHO Director General Lee Jong-wook. Synthetic biology offers great opportunity for biosensors, however current designs require hours before useful output. To tackle this issue in the field, it's crucial that our project can respond in minutes, hence we have engineered a fast, modular sensor framework. This allows detection of a range of different parasites, and may also be used as an environmental tool for mapping their spread. We have developed two new technologies that enable our modular input/output - a novel cell surface biosensor, customisable for specific parasitic proteases, linked through quorum-sensing to a new 'fast-response' module capable of producing a detectable output in minutes. To demonstrate the concept, we've designed and fabricated B. subtilis to give a striking colour readout upon detecting the waterborne Schistosoma parasite which affects 200 million people worldwide.

Team Indiana: Plant Time Machines

Because of their 3D structure, multiple organelles, dynamic environmental responses, and other unique properties plants make interesting synthetic biology platforms. To demonstrate one application of power of plants in synthetic biology and introduce plants to iGEM, we aim to create plants that initiate different gene programs dependent on the time of day. To build off of current Biobricks, we chose to design plants that smell like wintergreen during the day and banana at night. We believe this can be accomplished by tapping into the promoter sequence of circadian regulated genes. In addition, we will also create an iGEM BBA 10 standardized plant transformation vector which can accept Biobricks and help deliver them to the genome of Arabidopsis.

Team INSA-Lyon: Droppy Coli : factory of PHB, application and improvement

Polyhydroxyalcanoates granules (PHAs) are universal prokaryotic storage compounds of carbon and energy. We aim to control their production in E. coli thanks to a new part: a strong promoter sensitive to the shaking speed and the temperature of the water bath. By controlling this production, our team focuses on two final purposes : (1) the granule as a storage system for overproduced lipids with medical applications, such as DHA or EPA and (2) the granule as self-cleaving micro-beads in order to purify a recombinant protein of interest. In bacteria, three separate monofunctional enzymes are required for PHA synthesis. In order to improve this pathway, we intend to model a single multifunctional enzyme based on the study of natural evolution of fatty acid synthesis in animals.

Team IvyTech-South_Bend:

Anyone who wants to enjoy bathing in natural bodies of water in or near areas populated by humans or livestock may encounter unsafe levels of enteric bacteria. Contemporary methods of assessing water quality have a slow turn-around time so we have taken steps to perfect a biosensor for rapidly indirectly quantifying the presence of enteric bacteria in natural water samples through the detection of quorum sensing factors. Previous IGEMS have exploited the LuxR/pLux system for the detection of a variety of N-acylhomoserine lactone autoinducers. We have taken steps to further perfect a biosensor based on this device by transforming a gram-positive bacteria host to eliminate any background autoinducer signal and to build-in an enzymatic “read-out” to obtain an analog output. We envision the development of a handheld monitor that uses this IGEM biosensor, immobilized on input paper strips, to rapidly detect unsafe levels of enteric bacteria in water samples.

Team Johns_Hopkins: Synthetic Voltage Sensitivity at The Transcriptional Level in Saccharomyces cerevisiae

If the goal of iGEM and the Parts Registry is to take the messy world of genetic engineering and transform it into something like the standardized world of electrical engineering, it may be useful if electronic systems could directly interface with biological systems. Past iGEM projects have used chemical or optical stimuli to actuate transcriptional responses. Our project, however, seeks to add voltage sensitivity to Saccharomyces cerevisiae. Baker’s yeast was chosen because in some sense yeast have a system that responds to voltage input. With a voltage stimulus one can open the voltage-gated calcium channels of yeast, causing calcium ions to rush into the cytoplasm. This causes calcineurin to dephosphorylate Crz1, which enters the nucleus and binds various promoters. Our group presents a library of characterized Crz1-sensitive promoters of both naturally-occurring and synthetic varieties. Genes downstream of these promoters are thus voltage-regulated in media containing calcium.

Team KAIST-Korea: DiscoverY: universal diagnostic yeast

Large portion of the world is still suffering from diseases despite of the availability of treatment -tuberculosis in Africa for instance. Such trouble originates from unavailability of cheap and effective diagnostic method. Team KAIST will present DiscoverY that is capable of diagnosing multiple diseases. S.Pombe chassis holds FGFR1-STAT1 pathway with modification in FGFR1, which becomes fusion antibody receptor in our system. When fusion antibody receptors on the surface come in contact with antigens, the pathway is initiated. The pathway ends with GFP expression as diagnostic display. The system will be tested with tuberculosis antibody, and simple replacement of antibody will make DiscoverY the universal diagnostic yeast.

Team KIT-Kyoto: E.coli Pen": Draw with your own color

Our team, KIT-Kyoto suggests an “E.coli Pen” as a new Art Tool. This brand-new pen uses no ink but medium in which genetically modified E.coli has been cultured. The Pen is able to express more than four colors in various intensities with single bacterial culture. This will be achieved by constructing plasmids carrying genes coding for four different fluorescent proteins under the control of seven promoters having different sensitivity to oxidative stress. The E. coli carrying these plasmids will produce different colors with various intensities by differentially responding to the gradient of hydrogen peroxide treatment. Different from previous passive BioArt in iGEM, the genetically engineered “E. coli Pen” provides an active and wonderful tool for us to purely enjoy the Art having a feeling for biotechnology.

Team Korea_U_Seoul: Heavy Metal Gang Captured By Capsule Cop

Toxic heavy metals such as arsenic, zinc, and cadmium in water are very harmful. Detecting these heavy metals is an important task. So we designed a heavy-metal-detecting E. coli. In order to design the system, we employed two fluorescence proteins (GFP, RFP) and aryl acylamidase as signal reporters. The aryl acylamidase converts a colorless acetaminophen(Tylenol TM) to a brown color substrate. Since the detecting E. coli has three heavy metal promoters, if more than two heavy metals coexist in a solution, the E. coli emit mixed fluorescence, so we simultaneously detect metals. Our goal is to synthetic modules put these three genes for different heavy metals in a row in E. coli and then utilized in the form of a lyophilized powder, which can be stored in a drug capsule to make it portable so that analysis of water is easily processed. We call it a "Capsule Cop".

Team Kyoto: The Fantastic Lysisbox

Genetic engineered cell death is imperative for biotechnological usage, such as bioremediation area. For controlling cell death, we designed “Lysisbox” consists of a pair of modules: “Killer gene” and “Anti-killer gene.” As the Killer gene for E.coli, we noted the lysis cassette [SRRz/Rz1gene] of λ phage coding for a holin and an endolysin. The holin forms pores in the inner membrane, and the endolysin access to and degrade the peptidoglycan by passing through the pores, leading the E.coli to death. As the Anti-killer gene, we chose SΔTMD1 coding for a dominant-negative holin that inhibits the formation of the fatal pores. The balance of these two genes expression level has a key of the E.Coli’s life or death. In addition, such controllable membrane pores must show critical functions for all living organisms with lipid membranes. “Lysisbox” will contribute a lot to future projects, thus you must say “FANTASTIC!!!”

Team Lethbridge: A synthetic biology based approach for bioremediation of the tailings ponds

The industrial methods, used to harvest the oil sands, produce contaminated water in the form of tailings ponds with many harmful chemicals such as naphthalic acids, catechol and heavy metals. We are targeting catechol for degradation into common metabolic intermediates of the Krebs Cycle by using xylE from Pseudomonas putida that codes for the protein catechol-2,3-dioxygenase. Catechol-2,3-dioxygenase is being targeted into microcompartments, formed by engineered Aquifex aeolicus protein, lumazine synthase, to reduce cross-talk and increase concentration. The complex will then be purified and applied to the tailings for catechol degradation. By funnelling other pathways through catechol we can develop efficient methods for the decontamination of the tailings ponds. Mms6 from Magnetospirillum magneticum removes heavy metals from solution by forming nanoparticles. The Mms6 protein will be secreted from the cell into the tailings for the removal of metals such as iron and cobalt for creating an efficient bioremediation process.

Team LMU-Munich: Production of azobenzene derivates in E.coli and selection of successful transformants by apoptosis

We are engaged in two projects: Project “Pathway” involves the creation of an artificial metabolic pathway for the synthesis of azobenzene derivates in E. coli. This would be accomplished by expressing the required enzymes, encased in a proteinaceous bacterial microcompartment. This construct is necessary in order to shield the cell from toxic intermediates which would otherwise make this biosynthesis impossible. Azobenzene derivates are interesting in the field of biochemistry because of their properties as synthetic molecular switches. Project “ApoControl” is divided into three subprojects on controllable cell-death. The goal is to develop a system to improve the efficiency and specificity of gene expression in eukaryotic cell-lines and more specifically, to select cells expressing the target gene against cells that do not. Here, proapoptotic genes instead of antibiotic resistance are used as a selection marker to induce clean cell-death at different stimuli.

Team Macquarie_Australia: Engineering a Bacteriophytochrome switch – creating a controllable E. coli chameleon

Photoreceptors are utilized by almost every organism to adapt to their ambient light environment. Our aim is to engineer a novel, reversible molecular ‘light switch’ within E. coli by introducing a photoreceptor from non-photosynthetic bacteria (Deinococcus radiodurans and Agrobacterium tumafaciens). By cloning the bacteriophytochrome coupled with heme- oxygenase, an enzyme producing biliverdin, the created colonies are able to respond to red and far-red light environments. This novel approach will result in the colour of E. coli to ‘switch’ from blue to green reversibly. Our E. coli chameleon will serve as a fundamental ‘bio-brick’ for future applications by providing a simple and photo-reversible switch.

Team METU_Turkey: E-CO Sensor

Cells can sense and respond to the presence of various gas molecules such as oxygen, nitrogen and carbon monoxide using gas sensor proteins. CooA is a carbon monoxide (CO) sensing transcription factor. It is a member of the cAMP receptor protein (CRP)/fumavate nitrate reduction (FNR) family of transcriptional regulators. CooA switches on oxidation enzymes in Rhodospirillum rubrum (a purple, nonsulfur, phototrophic bacterium) which enables the bacterium to use CO as a carbon source. CO is an odorless and colorless gas which can be extremely lethal. Our aim is to develop a cell sensor which can detect a wide range of CO concentration in the environment. We are building CooA and CooA-responsive promoter biobricks which will be transformed into E.coli. Fluorescent proteins (GFP and RFP) will be utilized as dose-responsive signals of ambient CO.

Team METU_Turkey_Software: BIO-GUIDE

Team Mexico-UNAM-CINVESTAV: A very cool E. coli

We begin by proposing a biosynthetic construction that enables Escherichia coli to produce an antifreeze protein, AFP at less than 15 degrees Celsius. This protein prevents ice crystal formation in the cell, which in turn allows survival at very low temperatures. We develop a switch by adapting the cold-shock E. coli operon with AFP from a fish (Macrozoarces americanus) using a positive feedback circuit. A very important potential application we are interested in is the use of AFP in designing systems helping crops to avoid potential damage from frosts. There are other possible important applications in tissue and organ preservation.

Team Michigan: Algae Bioflocculation for Biofuel Production and Bioremediation of Oil Sands Tailings Water

Our team worked on two projects this year. Our first project aims to improve the economics of algal biofuel production by creating a cost efficient microalgae bioflocculant out of E. coli. To achieve this, we over-express Type I pili to increase the cell’s adhesiveness, and also express a chlorovirus protein on the cell surface which specifically binds Chlorella species, a promising algal feedstock for the biofuel industry. We are also participating in the Oil Sands Initiative and seeking to improve the biodegradation rate of naphthenic acids (NAs), a toxic by-product of the oil extraction process which can linger in the environment for decades. Two Pseudomonas strains have been found to synergistically degrade 95% of NAs. Our project focuses on engineering these Pseudomonas strains to form biofilms in the harsh tailings water environment, which can potentially increase degradation rates by two orders of magnitude, by expressing a self-associating E. coli protein.

Team Minnesota: _

Team Missouri_Miners: The Electric Microbe: Making A Fuel Cell With E. coli

The growing need for alternative fuel sources has sparked interest and research across many scientific and engineering disciplines. The fledgling field of microbial fuel cell development has previously relied on anaerobic metal reducing organisms such as Geobacter sulfurreduccens. This project sought to isolate genes from the electron shuttling pathway in Geobacter and transform them into the more manageable aerobic Escherichia coli. The Missouri University of Science and Technology iGEM team isolated four outer membrane cytochrome (omc) genes from Geobacter, vital to the extracellular transportation of electrons. The four genes; omcB, omcE, omcS and omcT, were cloned into individual plasmids. The eventual goal is to combine all four genes into one plasmid to transform into E. coli to create an aerobic, electron transporting microbial system.

Team MIT: Programmable, Self-Constructing Biomaterials

Our goal is to produce adaptive, living biomaterials that can be reliably controlled in two different systems: mammalian cells and bacteria. Our mammalian system uses newly isolated mechano-sensing promoters and a bi-stable toggle to stimulate osteogenesis via transient mechanical signals. Our bacterial system uses a toggle that takes advantage of quorum sensing and cell response to UV light and triggers the production of fluorescent proteins, and a polymer composed of a matrix of cross-linked phage. Our systems are remarkable because they translate a macroscale input into a pattern that emerges from the growth and re-modeling of cells. This technology not only has applications in the field of self-repairing nanotechnology and medicine, but it is also shedding light on artificial differentiation and the use of phage display technology in a new and innovative way.

Team Monash_Australia: Design and construction of a biological ethylene generation device

The Monash University iGEM team has identified that ethylene, a common organic compound, is under increasing production demands by the plastics and food industries. Current methods of production are energy intensive, and rely on processing of non-renewable fossil fuels. However many plants produce ethylene from L-methionine by use of the Yang cycle, which has lower energy requirements. We aim to introduce the genes that are required for ethylene production into Escherichia coli under the control of an inducible promoter, in an attempt to develop a cleaner and non-energy intensive method of production. At lower yields, this device may also provide a useful module for signal transduction between the E. coli and plants.

Team NCTU_Formosa: Mosquito Intelligent Terminator, a genetically engineered, temperature controlled E. coli for killing wrigglers

The Mosquito Intelligent Terminator (MIT) is designed and optimized to be an ecological and environmental friendly mosquito pesticide. MIT is an engineered E. coli secreting crystal proteins isolated from Bacillus thuringiensis to kill mosquito larvae, or known as wrigglers. These crystal proteins are toxic to certain types of mosquitoes and are not pathogenic to mammals. We designed a temperature-dependent genetic circuit expressing high levels of crystal proteins at room temperature only, thus production does not occur at incubation temperature 37°C. In order to make an environmentally safe insecticide, our design also incorporates a genetic circuit controlling the population size of E. coli. This intelligent terminator is not limited to mosquitos, as it can be custom fitted with different cry genes to other insect species. Currently, with more than one hundred crystal proteins targeting various insect species, our design may potentially serve as a promising pest control solution in the future.

Team Nevada: Development of Plant Biosensors for Environmental Monitoring Using Nicotiana tabacum Protoplasts as Transgenic Plant Models

The 2010 Nevada iGEM team has three objectives for this year’s competition. One, we want our highlight to be the first team to provide the iGEM registry with stress-inducible promoters to be used in plants. These promoters can be valuable tools in monitoring the environment for salt, heavy metals, temperature, and more. Second, we want to develop a real-time monitoring model of these stress-inducible promoters by having fluorescent reporters linked to their expression. Current research typically uses microarray, a technique that takes a ‘snapshot’ of a system, where as we want to hold a ‘video camera’ up to specific genes. Third, we will show the advantages of using Nicotiana tabacum protoplasts (NT cells). Our NT cell system provides a faster, cheaper, and safer method of obtaining a transgenic plant model than transforming an actual plant, benefits future iGEM teams may want to take into consideration.

Team Newcastle: BacillaFilla: Filling Microcracks in Concrete

BacillaFilla, an engineered Bacillus subtilis, aims to repair microcracks in concrete, which can cause catastrophic structural failure. BacillaFilla would be applied to structures by spraying onto their surface. The Bacillus swims deep into the microcracks. Repair is effected by production of CaCO3, filamentous cells and Levansucrose. CaCO3 expands at the same rate as concrete, making it the ideal filler. A filamentous cell mesh provides reinforcement. Levansucrose glues CaCO3 and filamentous cells in place. B. subtilis 168 sporulates, making it ideal for storage and transportation. The cells are naturally tolerant to concrete's high pH. We repaired 168's defective swrA and sfp, regaining motility. At the end of the crack the quorum communication peptide subtilin triggers a co-ordinated population response from a subtilin-inducible promoter. Upregulating SR1 and rocF promotes arginine and urea production, increasing exogenous  CaCO3 deposition. Over-producing  yneA induces  the filamentous cell phenotype, while SacB converts extracellular sucrose to levansucrose glue.

Team Northwestern: SCIN - Self-regenerating Chitin INduction

Chitin, found in the exoskeletons of insects and crustaceans, is one of the most abudant substances in nature. Like keratin in skin, it comprises the protective outer layer of these animals. Our chitin expression platform involves generating a layer of chitin from a lawn of bacteria in response to an external molecular cue. This cue induces chitin synthesis (fast) and cell lysis (slow). This system allows for a build-up of chitin followed by cell lysis and subsequent release into the top layer of the lawn. Abrasions expose cells to the external cue for self-repair. In this way, we create a regenerative chitin biolayer with potential medical and industrial applications.

Team NYMU-Taipei: SpeedyBac

Team NYU: ImmunoYeast : antibody discovery and production in one simple system

The goal of our project is to increase the speed and efficiency of the antibody discovery process. We constructed a yeast strain that is capable of screening a library of antibody fragments against an antigen of interest, processing the antibody genes through recombination and secreting an easily-purified form of antibody protein for research use. Our hope is to demonstrate the feasibility of using the yeast cell to not only discover antibodies but to provide a streamlined processing unit that can quickly and easily transition from antibody discovery to protein production.

Team Osaka: Continuous Greening Cycle

Desertification all over the world causes famine, drought and suffering. We aim to develop micro-machines that can stop and even reverse desertification by recovering vegetation in these areas. We envison a ‘Continuous Greening Cycle” in which engineered microorganisms decompose plant fibers into nutrients through the action of cellulolytic enzymes. They then produce water-absorbant polymers such as poly(gamma-glutamic) acid that retain water in the soil to help plants grow. When the plants die they will be decomposed to start the cycle anew. In addition to aiming for the continuous and self-expanding greening of desert areas, we hope to contribute to iGEM by developing useful BioBricks!

Team Panama: _

Team Paris_Liliane_Bettencourt: Every bacteria counts!

Counting is the action of finding the number of elements in a set.  Past attempts at developing counters in cells have mostly attempted to mimic the binary methods that computers use to count.Our first counter takes a new approach to counting in cells, essentially a mechanical rotary counter implemented on a micro scale.  Each time the counter detects an input, it performs an excision and integration directly down-stream of the active site, turning on a reporter and rotating over one "notch" on the counter.Our second counter operates on the wholly different principle that the statistical occurrence of a rare event in a large population can be modeled.  Each cell in our population harbors a construct that when stimulated has a small chance of excising a terminator and expressing a resistance gene.  The number of resistant cells is thus an accurate count of the number of input stimuli.

Team Peking: _

Team Penn_State: _

Team Purdue: Development and Characterization of Hypoxic Stress Response Systems in Mammalian and Plant Models

From water-logged soils to overpopulated regions of tumors, low-oxygen environments distress plant and mammalian systems. Plants with inadequate levels of oxygen move from aerobic respiration to alcohol fermentation to sustain their metabolism. This switch causes the accumulation of byproducts that are detrimental to the plant. A synthetic biological circuit, centering on the alcohol dehydrogenase (Adh) promoter, has been developed indicating when low oxygen levels (< 5% O2) are present in plants. Similarly, low oxygen zones can develop in solid tumors in numerous mammalian cancer models. Substantial evidence indicates that hypoxia in tumors initiates angiogenesis, a process that aids in tumor proliferation. Accordingly, an additional hypoxia-sensitive circuit that up-regulates the activity of a reporter protein in low oxygen (<1% O2) environments has been created for mammalian systems. The development and characterization of these circuits will provide tools to explore the consequences and identity of hypoxic environments in mammalian and plant systems.

Team Queens-Canada: WormWorks: Introducing the nematode C. elegans as a multicellular chassis

Historically, the iGEM competition has tended away from working with eukaryotic and multicellular organisms, limiting prospects for higher levels of project complexity in favor of simpler and easier-to-understand bacteria. The nematode worm Caenorhabditis elegans was examined as a prospective chassis for use in the competition. Once it was decided that the opportunities presented by the organism appeared to outweigh the challenges involved in working with it, a foundational library of parts was built and tested within the organism. This collection includes useful promoters, reporters, effectors, and a terminator. An educational resource specifically targeted at iGEM participants was written and incorporated into the team wiki in order to assist future teams in learning about and exploring the possibilities offered by C. elegans.

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _

Team _: _