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From 2013.igem.org

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Team AHUT China: Shining Sanctifier

Water, the origin of life, is the necessary and elementary component of our daily life. Various kinds of means have been developed to dispose nitrite and ammonium which are the main contaminants of this type of effluent. One of them is anaerobic ammonium oxidation bacteria (anammox) which can convert the fomite in the water into nitrogen. Our goal is to design a wastewater treatment system which can absorb the pollutant efficiently while transform it into luminous energy. We plan to use E.coli to design a bacterium that can digest the nitrite and ammonium in its interior using the disposal system from the anammox. Through the introduction of luciferase, the energy can be transformed into bioluminescence. Therefore, we named it Shining Sanctifier. This new star in synthetic biology will be applied to the sewage treatment system on a large scale while it can also be made into illuminating system.

Team AITM-Nepal: siRNA MEDIATED IMMUNE MODULATION FOR INNATE AND ADAPTIVE RESPONSE USING GENETICALLY ENGINEERED Escherichia coli

Canonical small interfering RNA (siRNA) duplexes are potent activators of the mammalian innate immune system. The induction of innate immunity by siRNA is dependent on siRNA structure and sequence, method of delivery, and cell type. The delivery of siRNA in a packaged outer membrane vesicle of gram negative bacteria is the theme of our work. The toll like receptor-7/8 activation by siRNA in order to boost the production of Interferon type -1 molecules to inhibit the viral and outer membrane LPS structure to activate Toll like receptor -4 to inhibit bacterial pathogens is the objective of this work. The delivery is made dependent on the peptide fragment which mediated the fusogenic mechanism so as to escape the endosomal compartment once endocytosed inside host(mamalian) cell. Thus freeing the siRNA to silence the myD88 transcript in host cytoplasm making RISC complex and hence, activating TLR-7/8 in endosomal membrane formerly.

Team BIT: A New Strategy to Detect Antibiotics in Milk: Based on Sensors with Controllable Bio-enhanced Blocks

Bio-amplification, especially controllable bio-amplification is significant for biological detection. In a synthetic biological way, 2013 BIT iGEM assembled the T7 RNA polymerase gene and T7 promoter as an amplification block (amplifier), which is based on the high activity of T7 promoter to amplify the signal. To make the magnification controllable, a lacO operator regulated by lacI was assembled in downstream as a control block (controller), by adjusting the concentration of IPTG. With this block, several sensors of materials including but not limited to antibiotics are able to be enhanced controllable. This year, a sensor of beta-lactam newly designed and one of tetracycline are applied to detect the residual of antibiotics in milk which endangers human health. To make the detection faster and more convenient, milk samples and engineered E.coli are mixed in a tailor-made bio-chip and the green fluorescence will be detected and shown on a tailor-made electronic equipment.

Team BIT-China: Intelligent Microbial Heat Regulating Engine

To keep the cells in a good condition, cooling system is used to control the temperature in fermentation process. However, the cooling system can result in a great consumption of energy, which increases the cost of production and causes resources wasting, global warming indirectly. To settle this problem, we constructed an Intelligent Microbial Heat Regulating Engine (I'MHeRE), which includes the customized thermo-tolerance system and the intelligent quorum regulating system, to help cells resist heat by regulating the expression of heat shock proteins and controlling the density of cells. The chassis host with I'MHeRE may make the fermentation less depend on the cooling system and shrink cost. Besides, cells could live well in higher temperature, because we extend their optimum living temperature and make them live in optimizing density. Owing to this, the activity of the enzymes in cells could be increased and the efficiency of microbial metabolism could be improved.

Team Biwako Nagahama: AgRePaper&E.coli-ink

Cellulose is used as raw material for paper, so our team experimented various ways to increase the amount of cellulose produced by agrobacterium and using it to make papers. For this we developed the different parts to insert into the system of agrobacterium. Among them are the genes used for expression of the curdlan. Similarly, genetic parts in order to increase the expression of the cellulose, along with the agrobacterium type binary vector were also developed . We are also working on recycling the produced paper by degrading the cellulose to D-Glucose using various enzymes. We worked for the preparation of the biological ink using the sperm whale's cells by genetically modification to increase amount of myoglobin. Then, we observed the change on the color of the product by altering the formation of myoglobin and the production amount of myoglobin with the insertion of T7 promoter to the cell system.

Team CAU China: Alcohol-detoxic Beverage

Alcoholism is prevalent in China. Here we decide to invent an alcohol-detoxic beverage that can considerably prevent alcoholism by adding one healthy bacterium-lactobacillus. In principle, this engineered bacteria can survive in the extremely acidic stomach environment and reduce the toxicity by converting alcohol to corresponding carboxylic acid through a two-step reaction. The two-step reaction is catalyzed by intracellular alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH),respectively.We try to engineer both enzymes, ADH and ALDH, to be acid resistant for higher performance in human stomach.

Team Chiba: Magnetic E. coli

In nature, there exist a variety of magnetotactic bacteria. Recently, it was reported that non-magnetotactic cells such as yeast can be magnetized to some extent. We set the goal to transform E. coli into those that are attracted by magnets. By magnetizing E. coli, the cell harvesting process will be much simpler and more economical than the conventional processes such as centrifugation and filtration. To this end, we are conducting three itemized projects. (1) modification of iron transportation network to import as much Fe ions as possible in E. coli, (2) sequestering/ storing iron into human ferritin, and (3) converting cytosolic space from reducing to oxidizing in order to elevate Fe(II)/ Fe(III) ratio within. Because all such manipulations significantly impact the physiology of the host cell, we are establishing the BioBrick platform that enables the temporal knockdown of multiple genes using recently control technology such as CRISPRi.

Team Fudan: ALeader: leading the advance of RNA synthetic biology

RNA regulation patterns, which have not been fully understood so far, is a research hotspot still deserving exploiting. A recently-discovered riboswitch ALeader updated our ideas by its delicate, 75nt-structure consisting of an aptamer, a recombination site, and even a bicistron motif. Inspired by this natural design, we proposed a series of novel strategies this summer, with dynamic rather than static perspectives. Guided by the theoretical study on functional multistable states and semi-static states of a riboswitch, and the kinetics involving impacts from other systems such as CRISPR, RNA polymerases, ribosomes, and degradation complex, the ALeader-based functional multi-phase and tricistron switches are designed. We also tried to regulate aptamer’s function by manipulating its working environment instead of itself, with SpinachALeader-based real-time monitors to avoid the signal distortion. Furthermore, to demonstrate the advantages of RNA biobricks, we constructed an antibiotic-detector with ALeader, optimized by a network with a RNA-OUT/IN translational regulatory system.

Team HIT-Harbin: B-POM: Biological proportional operational Mu-circuit

The composition of B-POM is that hrpR's promoter depends on the input, but hrpS’promoter is always Ptet and tet owns PhrpL, while the output gene follows tet and shares PhrpL. Once the input is sensed, the input promoter triggers hrpR's transcription. The activity of Ptet is constitutive, which means HrpS protein is ample. As HrpR accumulates, HrpS binds to HrpR and form HrpRS which then triggers PhrpL, and tetR and output begin to accumulate. tetR can inhibit Ptet. As a feedback, HrpS and HrpRS will decrease. PhrpL will be of lower activity, so the amount of tetR and the level of output will decline. The decrease of tetR will enhance the hrpS' expression. All these construct a feedback cycle. Finally, the output will stabilize and be in a certain proportion with the input. By manipulating the RBS of hrpR, hrpS, tetR and output gene, we can control input-output proportion.

Team HokkaidoU Japan: “Maestro E. coli” ~optimization kit for expression~

Thousands of genes are expressed in living cells. Their expression is cleverly controlled by promoters and RBSs. Precise regulation of recombinant genes is hard to achieve. Imbalance in regulation results in little production. However, it is hard to objectively select promoters and RBSs. We thought that E. coli could do the selection for us. We created a kit for E. coli to find the best suited promoters and RBSs. It enables our lab E. coli to be like “Maestro” who creates excellent harmonies with lots of instruments. For the kit we created an original promoter and RBS families with different strengths. We checked and made these parts to be reliable. And it only takes a single golden-gate assembly to get your construct! We made the promoters and RBSs by selecting from randomized libraries. Using the kit, E. coli can choose optimal promoters and RBSs by her/it/him-self, just like the maestro.

Team Hong Kong CUHK: Switch off PAHs

To rapidly regulate biological process, we designed a novel transmembrane protein called Voltage Switch (VS), which is a fusion protein utilizing the voltage sensing domain from potassium ion channels. Triggered by change in potential across the cell membrane, VS can separate or bring targeting enzymes into proximity, thus allowing an instant control of enzymatic reaction. We also utilized VS to accelerate the polycyclic aromatic hydrocarbons (PAHs) degradation system – another highlight of our project. The metabolites of certain PAHs are mutagenic and carcinogenic. We codon-optimized laccase from Bacillus sp. HR03 and catechol 1,2-dioxygenase from Pseudomonas putida KT2440 for Escherichia coli, which when forming a cascade, PAH degradation into less toxic simple carboxylic acid would occur. Since quinones are intermediates in the degradation of PAHs, we also added quinone sensing and response repressor (QsrR) to control the degradation process.

Team Hong Kong HKU: E. capsi: Reducing phosphate pollution using engineered E. coli that harvests polyphosphate

Phosphate pollution in waterways and water treatment plants is a major problem. Removal of phosphate from wastewater is required to treat phosphate-containing discharge to reduce eutrophication, algal blooms and “dead zones” in lakes, rivers and coastal marine ecosystems. The aim of this project was to remove or reduce the levels of inorganic phosphate from a system or environment by employing engineered bacteria E. capsi, capable of accumulating phosphate in the form of polyphosphate. Our strategy is to express polyphosphate kinase together with the ethanolamine utilization (eut) bacterial microcompartment from Salmonella enterica to provide an environment for polyphosphate synthesis. Furthermore, the project provides a novel way to recover accumulated polyphosphate, an energy rich macromolecule with many industrial uses. This paves a way towards living system-based phosphate pollution treatment to tackle critical environmental challenges.

Team Hong Kong HKUST: FATBUSTER - The Artificial Futile Cycle

While low-fat diet and regular exercise are popular approaches to fight with obesity, one easy alternative is simply to increase energy metabolism. In a synthetic biology approach, we are working to create an artificial futile cycle in mammalian cell by introducing glyoxylate enzymes native to bacteria. Past research has shown that mice expressing enzymes constituting an active glyoxylate shunt are shown to be resistant to diet-induced obesity. Our team plans to introduce an inducible system that allows us to couple the sensing of circulating fatty acid concentrations with an inducible circuit of glyoxylate shunt. Our inducible system is intended to prevent the risk of fatty acid deficiency, while facilitating greater fatty acid uptake at higher fatty acid circulating concentrations. Such a system should increase the feasibility of a glyoxylate cycle engineered to function in vivo.

Team HUST-China: Antihypertensive Ecoli

Hypertension causes grave concern worldwide for its notoriety, there’re not many therapeutic methods to hypertension besides various antihypertensive drugs. However, this comes along with heavy financial burden to developing or underdeveloped countries. In addition, almost all these drugs have side effects to liver and renal. Here is a novel method to treat Hypertension by constructing human-friendly engineering bacteria that can produce short-chain fatty acids (SCFA) periodically and naturally to help maintain the blood pressure in safe level. SCFA, especially acetate and propionate, has been proved to induce vasodilatation and ensuing hypotensive response via receptors in smooth muscle cells of vessels. This year we have found a metabolic pathway in Escherichia coli that converts succinate to propionate through Wood-Werkman reaction. An operon consisting four genes encodes enzymes in this pathway. By combining bio-oscillator and key gene together, we want to make E. Coli release propionate periodically in patients’ intestine periodically.

Team HZAU-China: Safe moving vaccine factory

For HZAU-2013iGEM project, we are creating a safe moving vaccine factory by synthetic biology which can spread Rabies vaccine in dogs rapidly and actively. Our aim is to help in the achievement of the WHO goal of being free of human rabies by 2020 through the improvement of the vaccination coverage in dogs. The idea comes from Yersinia pestis and fleas. We make use of fleas as our moving injector. When flea feed blood from dogs, our vaccine vector Bacillus subtilis will be regurgitated into blood and successfully transferred to mammalian host. Bacillus subtilis can express antigens which can stimulate the immunity of dogs. Meanwhile, endogenous or exogenous expression of 'Antimicrobial Peptides' by B. subtilis can kill Yersinia pestis in fleas. In this way we achieved a safe moving vaccine factory.

Team IIT Delhi: pHColi

pH induced response elicited by certain promoters in bacteria may have major practical applications. The response can be targeted for specific pH ranges, for example in tracking the anomalies associated with the gut micro-biota or detecting pH inside a bioreactor. There are only limited studies reported in the area. In the present project, a genetic circuit has been created, using the promoters of the acid shock response gene from E.coli and the F0F1 ATPase operon from C. glutamicum that produces a pH dependent colour gradient, much like a universal pH indicator. A mathematical model has been developed to simulate the experimental findings. The present study will form the basis for further research in the field of synthetic biology.

Team IIT Madras: COMBATING SHIGA TOXIN : A SYNTHETIC BIOLOGY APPROACH

Shiga toxin, a worldwide menace, has killed over 1 million people to date and continues to afflict almost 150 million people each year. Currently, there is no treatment for Shiga toxicosis and it leads to complications in the human system like hemolytic uremic syndrome (HUS) and renal failure. Here, we propose a two-fold, novel synthetic biology approach to combat the lethal effect of the toxin. We aim to neutralize the already produced toxin through a nine amino acid Gb3 mimic peptide. We have engineered the Gb3 mimic along with a cellular export signal (ompF) downstream of AHL(quorum sensing molecule) inducible promoter (pLuxR). We also plan to prevent further toxin production by inhibiting the biofilm formation of shigatoxigenic E.coli using indole-3-acetaldehyde (I3A). We expect to validate our approach through functional assays and in silico modelling. Our findings can potentially initiate a new perspective of tackling Shiga toxicosis using synthetic biology tools.

Team ITB Indonesia: Aflatoxin Biosensor

Aflatoxins are naturally occuring mycotoxins that are mutagenic and carcinogenic. Aflatoxin contamination of foods that are found in many developing countries may cause a serious problem for human health. ITB_Indonesia team for iGEM 2013 focuses on designing a whole cell biosensor for aflatoxin B1 detection in foods. The biosensor uses Escherichia coli as the chassis to build a genetic circuit using SOS response system to detect DNA damage caused by aflatoxin B1-oxide attack. The SOS response promoter is followed by a reporter gene coding a chromoprotein, therefore the concentration of aflatoxin B1 in food samples could be easily detected by the color change of the bacteria. For the ease of usage, we will design a syringe shaped device with our whole cell biosensor in it. This device would allow aflatoxin B1 to enter the device, but would not permit the cells to leave the device.

Team KAIT Japan: Hay fever curE.coli

Japanese on of six people is troubled now by hay fever. These people take a medicine for the hay fever. But, If they take it , they become sleepy. If become sleepy, they cannot work and study. So, we are working on a project to relive hay fever by Escherichia coli to improve these. Mechanism of hay fever When an allergen invades it in the living body, naïve T cell differentiates in Th2. There is more Th2 than Th1, and the mast cell and others that is humoral immunity become active, and inflammation is in this way caused. We perform following four this time. ①Expression of IL-10 receptor to E.coli. ②Phosphorylation of STAT3. ③Preparation of gene array with HlyA and L-12 promoter and receiving the STAT3. ④Preparation of gene array with TolC and HlyB and HlyD promoter and to receive the STAT3.

Team KIT-Kyoto: Fregrance coli

We are trying to construct a novel E.coli that has fruity flavor like Japanese rice wine (Japanese sake). In order to accomplish the purpose, yeast genes related with production of the Japanese sake fragrance were introduced into E. coli cells. We also tried to develop a way to eliminate bad smells of E. coli in parallel. Although we previously won a gold prize by the development of a novel pen (E. coli Pen) in 2010, its bad smells were weak points and must be improved. We will overcome this problem through the progress of our new project in 2013. So far, “smell” is not a popular keyword and not a major field in iGEM. However, we believe that our project will provide a new point of view to iGEM friends

Team Korea U Seoul: Pearl-coli: E. coli converting CO2 into a pearl powder (nacre)

The Korea_U_Seoul team aims to design Pearl-coli that is E. coli able to convert atmospheric CO2 into pearl powder materials. The design is based on cell surface display of nacrein in E.coli. Nacrein is a major protein component in nacre(an organic-inorganic composite layer found in outer coating of pearls). We divided nacrein into functional regions - carbonic anhydrase(CA), calcium binding and scaffold repeats. CA domain fixes CO2 into carbonic acid changing to bicarbonate ion in aqueous solution. We will examine if displayed nacrein in E. coli can make a pearl powder in a solution or fabricate a nacre-like structure while atmospheric CO2 is fixed into bicarbonate. Once a nacre material can be prepared from Pearl-coli, we will grow E. coli in a confined container to make synthetic pearl. The Pearl-coli has dual-function such as (1) mitigate the global warming by CO2 reduction, (2) prepare valuable pearl-like raw materials.

Team Kyoto: Oscille.coli

Every organism has its own cycle such as the periodicity of cell division, ordered patterns of its body. Some kinds of the cycles are regulated just by two factors. Using E. coli, we applied this kind of periodicity formation.</p> Firstly, we focused on oscillation regulated by RNA. We suspected if RNA world hypothesis is correct, there could be protein-depended oscillatory system. To show the possibility of cycle formation by RNA, we constructed an oscillator by utilizing two different types of functional RNA, which are transcriptional activator and repressor.</p> Secondly, we also targeted on planar oscillation forming mechanism. A. Turing (1952) suggested a simple principle containing just two variables explains many organisms’ epidermal pattern formation. However, it is not confirmed the pattern formation is only based on Turing’s discourses. To check this, we used two types of E. coli, which secrete different factors, and regulated their population.

Team LZU-China: Twinkle Cancer Hunter

To construct a regulating vector of NF-κB signaling pathway by gene recombination technology, introducing into tumor cells with NF-κB to form a signal feedback control system. Using NF-κB binding elements as promoter,and IκB-GFP fusion protein as reporter.Then inverted into HEK-293T cells and DU-145 cells.Through the observation of the GFP to probe the expression of IκB. The expressed protein was identified by Western blot, etc.The constructing of a regulating vector of NF-κB signaling pathway provides a new method and thought for tumor gene therapy, and propel forward the research of NF-κB signaling pathway.

Team Macquarie Australia: Green is the new black - Expression of Chlorophyll within Escherichia coli

Photosynthesis is a key biological pathway that uses sunlight energy to convert water and carbon dioxide into ATP, glucose and oxygen. Chlorophyll is a green pigment that facilitates this energy production in photosynthetic organisms. Although the biosynthesis pathway for chlorophyll has been thoroughly investigated, the reproduction of this pathway in a non-photosynthetic organism has, to date, not been achieved. Successful production of chlorophyll in a bacterial host is the first step towards the synthetic construction of photosystem II, and the eventual creation of a renewable energy source. Our research involves expression of twelve genes (from Chlamydomonas reinhardtii) necessary for the chlorophyll biosynthesis pathway in a bacterial host (E. coli). Gene sequences have been synthetically designed to allow for prokaryotic expression. By utilising Gibson assembly, we plan on being able to successfully produce chlorophyll in prokaryotic cells. This will be evident from the growth of green E. coli colonies.

Team Nanjing-China: Atrazine Elf

Atrazine, a widely used herbicide, persists for a long period in the environment onced used. It causes metabolic disorders in both animals and humankind. Our team utilized the ribosome switch induced by atrazine, a QS system of Plux and a degrading enzyme to control E.coil’s motility through regulating it’s CheZ gene. Therefore, E.coli can recognize atrazine, recruit team workers, and degrade atrazine. Our team found a transporter of atrazine, which we call TRM. We also mutated the degrading enzyme, TrzN, making it better at degradation. We combined TRM and the TrzN to improve atrazine absorbance and degradation. Moreover, our team are trying to analyze and compare several systems with computer, hoping to find the best one which is equipped with faster moving and quicker degrading. Overall,we believe our system will boost the industrialization, universalization as well as standardization in the field of treatment for atrazine and other versatile small molecules.

Team NCTU Formosa: E.colightuner

We have proven a sRNA-regulated system of our own to be an effective and competent way for regulating gene expressions. Recent studies have shown that sRNA-mediated regulation is an important factor to bacterial growth. sRNAs work by base pairing with limited or extended complementary target mRNAs, regulating protein productions. Using sRNA mechanism, we can control gene expression in RNA level, in contrast to common promoters that functions on DNA level. Since the existing sRNAs in Escherishia Coli have important functions in other metabolic processes, we designed an artificial sRNA with high specificity to avoid undesired base binding in vitro. By using the sRNA-regulated system, red light induced operator, and thirty seven degree Celsius ribosome binding site (RBS), we constructed a manipulatable system that is capable of expressing four different genes under different conditions. In other words, it is a multitask machine.</p>

Team NJU China: Biomissile: a novel drug delivery system with microvesicle

Recently, small interfering RNA (siRNA) has emerged as a promising therapeutic drug against a wide array of diseases. However, site-specific delivery has always been a challenge in gene therapy. Microvesicles (MVs) are lipid-bilayer vesicles which are naturally secreted by almost all cell types, playing crucial roles in intercellular transport of bioactive molecules. Given the intrinsic ability to naturally transport functional RNAs between cells, MVs potentially represent a novel and exciting drug carrier. In our project we are trying to express both anti-virus siRNA within the cell and target protein on the surface of the MVs by engineering the HEK 293T cell, which is capable of producing large amounts of MVs. Thus, the MVs produced by our engineered HEK 293T cells will contain the siRNA and be able to specifically deliver the siRNA to the sites we want, acting as biomissile for the targeted destruction of the disease.

Team NJU NJUT China: The Application of Cas9 as a Gene 'Missiles'

Most bacteria and archaea can resist invading DNA and/or RNA elements via the clusters of regularly interspaced short palindromic repeats (CRISPRs).It is believed that the integrated CRISPR sequences have the ability to form a genetic memory which prevents the host from being infected.The memory exist as a DNA library in genome, artificially modified to set its target. The CRISPRs and Cas (CRISPR-associated) interact and form this prokaryotic adaptive immune system. Cas9, as a core of CRISPR system, can play a role of targeted-attacking gene 'missiles'. Therefore, we build a sort of plasmids, loading CRISPR system, to realize the 'killing' of harmful genes and/or organisms.

Team NTU-Taida: QS array

Bacterial infection is the invasion of the body by pathogenic bacteria, which causes pneumonia, urethral infection, bacteremia and other symptoms in hospital and community. The efficiency of traditional detection and diagnostic approaches is impeded by the time-consuming laboratory procedures, yet many of which grow poorly in bacterial cultures. All these limitations call for a new rapid and direct bacterial identification method to improve patient management and antimicrobial therapy. Quorum sensing is a type of bacterial cell-cell communication correlates with the population size. Many bacteria have one or several species-specific quorum sensing molecules released in different growth state and environment. Quorum sensing signals are shown to be involved in many physiological functions, including virulence, biofilm formation and drug-resistance. We aim to establish a novel bacterial identification method in clinical samples based on the quorum sensing profiles.

Team NTU Taiwan: YeasTherm - against the cold

During winter season, due to low temperatures, fish farming is one of the most heavily affected economic venues. Due to this, year after year, several farmers are faced with many problems as a result of a loss of fish product. </p>Using our background in bioengineering we suggest an innovative alternative: Our idea is based on heterologues expression of SrUCP in Saccharomyces cerevisiae and Rhodotorula glutinis. Through the expression plasmid, yeasts are transformed from the wild-type phenotype into a thermogenic phenotype. </p>To implement this idea and make it simple and efficient, we plan to drive the expression of SrUCP under the control of cold shock promoter Tir1. In this way, yeasts will generate heat only when the temperatures drop. Moreover, the temperature-responsive range of Tir1 may be regulated by applying genetic circuits, providing the means to manipulate the biological device to suit different temperature conditions and needs in application.

Team NU Kazakhstan: Detection of Carcinoembryonic antigen with sandwich-biosensor

Carcinoembryonic antigen (CEA) is the cancer biomarker at early stages of several cancers including colorectal carcinoma, lung carcinoma and others. The aim of the study is to develop a biosensor that can be used in the detection of CEA. In the first part of the study ssDNA aptamers, with strong affinity for CEA, were selected by 12 cycles of Systematic Evolution of Ligands by Exponential Enrichment procedure, and characterized with dot-blot analysis and Surface Plasmon Resonance methods. In the last part, it is planned to clone the genes that will assist in expression of streptavidin on the surface of E. coli and S. cerevisiae membrane. E. coli will deliver streptavidin on the surface via Lpp-Omp expression system, while S. cerevisiae via Aga1 – Aga2 system. Modified model organisms, aptamers and CEA will be used to construct sandwich-biosensor.

Team NYMU-Taipei: Bee. coli: to bee, or not to bee

To save bees from Nosema ceranae, the culprit of colony collapse disorder, we created Bee. coli. from E. coli K-12 MG1655, a bacterium residing natively in bees. Bee. coli is strategically designed to work as follows. First, it continuously secretes mannosidase to inhibit the sprouting of N. ceranae spores. Second, if the bee is infected, the fungus-killing-circuit with a positive feedback design will be turned on to wipe out N. ceranae. Third, if these designer weapons should fail to conquer N. ceranae, our designed bee-suicide-operon will be activated to kill the infected bee and save its companions. Fourth, a light-inducible lysis system is included to ensure our Bee. coli only lives inside of the bee. Fifth, we apply encapsulation as the way to send Bee. coli into the bee. Since the capsule will only dissolve in a bee’s gut, our Bee. coli will not spread to the environment.

Team Osaka: Beat the discrimination against E.coli !

Since the middle of the 20th century, Escherichia coli(E.coli) have made great contributions to various field of our society. Although they have played essential roles in the society, it seems that they are not properly appreciated by general public. People's common images to E.coli are very negative (dirty, stinky, dangerous etc). So in our project, to wipe away the negative images to E.coli, we have created a circle that enable them to communicate with each other via nutrient production.And we made “empathetic E.coli” that lives cooperatively with each other. Then, by conducting experiments and using computer simulation, we have examined how they live and grow in liquid medium culture and what kind of pattern they form on solid medium culture.

Team OUC-China: Reconstructing the Magnetosome Membrane in E. coli

Membranous organelles are unique structures of eukaryotic cells, rare bacteria and paleontology. Magnetospirillum magneticum is an important biological model system of prokaryotic organelle study because the structure of magnetosome in Magnetospirillum magneticum has similar traits to eukaryotic organelles with membranes. Our task is to reconstruct the magnetosome membrane in Escherichia coli. Magnetospirillum magneticum requires a micro-aerobic and oligotrophic environment in order to produce magnetosome, so the significance of our project lies in simplifying the magnetosome produce method, opening up the path for futher functional gene research. We use homologous recombination to transfer the mamAB gene into E.coli to build an IMS part. Also, as the mamK gene is crucial to the IMS construction. We want to improve the mamK gene's expression by stabilizing its mRNA with a new method, hoping it can be used to promote the IMS construction. So we design a DNA segment to slow down mRNA degradation.

Team Peking: Aromatics Busted

Aromatic pollution is becoming a worldwide concern, and monitoring aromatics remains challenging. Noting the abundant genomic data of prokaryotes from aromatics-rich environment, Peking iGEM applied part mining to the genetic repertoire to develop a comprehensive set of biosensors for aromatics. The transcriptional regulators for each typical class of aromatic compounds were bioinformatically determined and promoter engineering and protein engineering were performed to tune their function. To expand the detection range, enzymes in upper pathways, working as plug-ins, were coupled with biosensors to degrade aromatics to detectable compounds. For environmental detection, we construct the band pass filter to detect a certain range of concentration. Responses of biosensors equipped with band-pass filter can robustly reflect the concentration of environmental samples. Peking iGEM has remarkably enriched the library of biosensors for aromatics and enabled quantitative detection for environmental monitoring. These biosensors will be also potent for metabolic engineering and well-characterized synthetic biological tools.

Team SCAU-China: Detection and degradation of organophosphorus compounds

Synthetic organophosphorus (OP) compounds, which are highly toxic contaminants in agro-environment and food security, have been widely applied to pesticides. Parathion is a typical representative of organophosphorus pesticides. This year, our goal is to construct a p-Nitrophenol sensor in E.coli, which is the degradation product of parathion, in order to reflect the existence of parathion. Besides, we try constructing a degradation system to solve the pollution problem. Considering the biosafety problem, we also design a suicide system in which the lethal genes are only triggered by declining p-Nitrophenol concentration. This will enable the bacteria to commit suicide when p-Nitrophnol is sufficiently degraded.

Team SCUT: E.cerevisiae

E.cerevisiae is a sophisticated signal transport system between E.coli and S.cerevisiae. Producer, the E.coli, is assigned to distribute a special volatile—butanedione periodically with a stable oscillation circuit, which defines the meaning of the signal. On the other side, Sniffer，the yeast, transplanted with a nose from nematode, can respond to the signal immediately. We hope this can realize the communication between prokaryotes and eukaryotes for the further research on symbiosis.

Team SCU China: Imitations of Gametogenesis & Sexual Reproduction using E.coli

We intend to construct two groups of differentiated E.coli,one imitates the male multicellular organism ,the other for the female. When cultured separately, the male/female multicellular system gets bigger and matures, and cells will differentiate into gametes,which cannot divide any more but are capable of gene transfer.</p> After that, you mix this two liquid cultures,the male gametes will recognize the female cells and begin to transfer modified F plasmids into female gametes through sex pili. The conjugation makes female gametes return to the state of un-differentiation(called G cells),which means they can divide again but are not sexually determined.Then,after several cell divisions,one G cell will differentiate into a G+ or G-,which, like zygote, can grow into next generation of the multicellular system maybe containing genes from both male and female gametes.

Team Shenzhen BGIC 0101: Genovo

Genovo is a Computer-Aided Design (CAD) tool used for denovo design of genome. The current version consists of 4 parts. The first, Chromosome Construction will grap genes in a common pathway and chromosome features to build a new genome and let user to define the order and orientation in drap-drop way. The second, Nucleotide Modification will optimize and soften the sequence of CDSs. It also help design the CRIPSR sites so that we can silence the wild type genes. The third, Chromosome Segmentation will cut chromosome into pieces and add 3A & Gibson & Goldengate & Homologous Recombination adaptors to the pieces automatically for assembly. The last one, OLS Design will guide users to gain the chromosome by microarray. Genovo will enable user to design their innovative chromosome as their wishes and further the research on genome on pathway level.

Team Shenzhen BGIC ATCG: Cell Magic

Cell Magic plays a gorgeous movie show in the both E.coli and S.cerevisiae.Various colors are blooming in different branchs & buds: plasma membrane, nucleus matrix, mitochondria membrane & matrix, vacuolar membrane, peroxisomal membrane, centrosome, and also actin. But the scene is far from static, colors will show up in order under the sophisticated cell cycle system at G1, S, G2 or M phase. Accelerator—degradation system is applied to run this movie faster, and freezer—sic1 system will put off the cell cycle during G1 phase. Beside, the editor—intron will expands a random dimension, leading to produce more combining form.

Team SJTU-BioX-Shanghai: Metabolic Gear Box

Few researches have been done to regulate gene expression levels in genomic scale so far. This year we aim to combine two systems together in order to provide a universal and convenient tool which can be used to regulate different genomic genes simultaneously and independently in a quantitative way.

Our project involves the newly developed gene regulating tool CRISPRi and three light-controlled expression systems induced by red, green, and blue light respectively. Simply by changing the regulating parts in CRISPRi system towards mRFP, luciferase, and three enzymes, we hope to prove our system can be used qualitatively, quantitatively and practically step by step.

We have also designed a box and written a software as our experiment measurements. Simply by typing in several parameters, different gene expression levels can be controlled. This system can also be improved to predict the maximized producing efficiency after some simple tests in future.

Team Sumbawagen: E. coli which able to measure the level of sugar in honey by emitting light

Glucose and fructose are major sugar component of honey. Sumbawa honey is protected as geographical indication by Indonesian patent office. Sode Lab at Tokyo University of Agriculture and Technology has created a fusion of mutant glucose binding protein and firefly luciferase, which able to measure glucose level by emitting light - intended initially for blood glucose sensor application (Taneoka et al, 2009). In this project, we plan to create this construct in Biobrick format, and evaluate the ability of transgenic E. coli for the measurement of glucose in honey. Our final goal is to create a device which can be used for quality control of Sumbawa honey, which we call 'ECONEY'.

Team SUSTC-Shenzhen-A: Game Theory--Stategy for the Classic Prisoners' Dilemma

There are many applications of the game theory in some aspects of our life. Each individual has two kinds of choices--to betray or stay silent, and the choice you make would determine your fate. To betray the other side, you may risk being revenged. While staying silent, companion's betrayal may hurt you deeply. As for our project, we work out a new way to imitate the game theory by constructing a community of two E. Coli bacteria. Here we use the growth rate of each species to represent its fate. The effect of one's silent or betrayal on the other species' fate is acted through intercellular signal molecules of two quorum sensing systems. Each signal molecule regulates the expression of toxic genes in the other species and reduces its growth rate. We characterize the consequence of each strategy by quantitatively measure the growth rates of each species in the community.

Team SUSTC-Shenzhen-B: Circuit+

To standardize genetic circuits and bring the idea that relations exist in circuit between parts back to synthetic biology, we proposed our Technical Standard RFC 101 and RFC 102 to define genetic circuits and logical gene gates. To solve the problem that synthetic biology lacks such a database to systematically record genetic circuits and to make the standards work, we built Circuit+, an online registry of standard genetic circuit which records information of circuits based on the two standards. Users can retrieve circuits, browse information, share by exporting SBOL and upload new circuits. We also have developed Clotho version Circuit+, Circuit List and transplanted TTEC to Clotho. And we have developed an online platform for synthetic biology lab management. We also did human practice to promote synthetic biology and iGEM.

Team SydneyUni Australia: Keeping DCA at Bay - Assembly of synthetic constructs and cassettes for degradation of dichloroethane.

The picturesque city of Sydney is marred by industrial efflux of chlorinated hydrocarbons into the aquifers around Botany Bay. 1,2-dichloroethane (DCA) is toxic and a suspected carcinogenic agent, and one of the more soluble and mobile contaminants. Conventional DCA treatment is both costly and time-consuming, involving pumping and heat-stripping groundwater. We propose a biological alternative which may be cheaper and more effective. There are strains of bacteria able to degrade low levels of organochlorine compounds in selective conditions. Polaromonas JS666 and Xanthobacter autotrophicus GJ10 contain two pathways of particular interest. Our goal is to construct our own versions of two metabolic pathways of DCA biodegradation for comparison in a BioBrick-compatible vector, and characterise their effectiveness in utilising DCA as a sole carbon source for growth. We hope to create friendly strains of bacteria capable of removing DCA at greatly reduced cost and effort, and reduce the environmental impact of industry.

Team SYSU-China: iPSC safeguarding Device

Since Shinya Yamanaka published the epoch-making paper in 2006, the induced pluripotent stem cells(iPSCs) has become one of the most promising techniques in regenerative medicine. Like embryonic stem cells(ESC), iPS Cells can be differentiated into any tissues. Compared with ESC, iPSC is easier to attain, immune rejection-free, and ethical issue-free. However, Rurther application of human induced pluripotent stem cells(hiPSCs) in tranlational medicine requires the concerns of two problems: the specificity of directional differentiation and the safety of the transplant. Here we design a new device which can spontaneously select hepatocytes from iPS differentiated cell mass and prevent potential carcinogenesis. To achieve accurate spatiotemporal control,we build a miRNA-122 sensor and make use of the tetracycline induction system. Our work may also be extended to the field of gene therapy, and provide a new direction to our train of thought about how to solve the safety problem in genetic manipulation of human cells.

Team SYSU-Software: CAST (Computer Aided Synbio Tool)- An Integrated Tool for Synthetic Biology

Accurate simulation and gene circuit design are essential but difficult parts in synthetic biology.Here, we designed CAST to cover the workflow from beginning to end, users can focus on function design and the gene circuit would be automatically designed. Furthermore, we developed a new simulation model that work with standard dynamic characteristic and verified by wetlab experiments. Moreover, we build an expandable database that users can contribute their own dynamic information which would lead to more accurate and sufficient dynamic information of all the Biobricks. Finally, our software is designed as an easy deployed server so that it can be used on personal purpose or shared by a whole lab or institution.

Team Tianjin: Alk-Sensor, a Novel Detector Applied for the Selection of Alkane Producers

Biosynthesized alkanes are promising candidates for drop-in replacement of petroleum. We constructed and characterized a device named Alk-Sensor, which can sensitively detect a wide range of alkanes and generate certain response. Alk-Sensor is composed of ALKR protein——a transcriptional regulatory protein, and promoter alkM. ALKR recognizes alkanes and their interaction triggered a conformation change of ALKR dimers which isomerizes the promoter-RNAP complex and led to activate the downstream genes of PalkM.

Based on Alk-Sensor, we built a relationship between productivity of alkanes with strain’s growth rate under certain environmental stress. Starting from this relationship we further designed a novel selection method to select out the engineered strains with highest productivity of alkanes. We demonstrated that this novel selection method could enable us to select out the optimized strains effectively and efficiently.

Team TMU-Tokyo: Genomic 'Pythagorean Devices'

In this year, TMU-Tokyo created Genomic 'Pythagorean Devices'. Pythagorean Device appears Japanese famous educational TV program 'Pythagorean Switch' Pythagorean Devices are known in the US as 'Rube Goldberg machines'. Pythagorean Devices are deliberately over-engineered or overdone machines that performs a very simple task in a very complex fashion, usually including a chain reaction. We constructed a Pythagorean device in Escherichia coli genome, using lambda phage recombination system 'RED'.

Team Tokyo-NoKoGen: Twinkle.coli -Fast cycle! Fast response!-

We created Twinkle.coli, which “blinks” fast　like a firefly. An oscillator is a system that responds in periodic changes. This response is usually regulated by positive or negative feedbacks by using inducer or repressor proteins. However, the use of proteins might delay the response because transcription and translation must happen before the next output. To design an artificial fast responding oscillating circuit, we designed an　RNA-based oscillator. We used RNA-responsive self-cleavage ribozymes whose cleavage is regulated by an RNA molecule. The ribozyme cleavage cuts-off an “RNA scaffold” that harbors RNA aptamers. This aptamer binds to its specific target proteins, which are directly fused to reporter protein. This binding recruits the already translated split reporter protein complementation resulting in the output (twinkles). Our system enabled fast response and short oscillation cycle.

Team Tokyo Tech: ‘Mutant Ninja. coli’

In our project, we propose to create E. coli that mimic some of the qualities of Japan’s ancient ‘ninja’ warrior-spies. A ninja must receive and pass on correct information at all times. A mistake will be fatal. We have created a circuit that avoids crosstalk between two signals in cell-to-cell communication, and we are also looking into applications for it. Ninjas are also known for their star-shaped ‘shuriken’ throwing knives. Our E. coli ninja has a similar weapon, an M13 phage which it releases to infect other E. coli, injecting plasmid DNA into them. Finally, ninja must harmonize with the natural environment, so their relationship to it is very important. Plant hormones help plants to grow efficiently, and we are attempting to construct a circuit that synthesizes two plant hormones depending on the soil environment.

Team Tsinghua: Mobile Health---Pathogen detector

In a long term, the testing of pathogenic diseases is via comparably complex procedures. This year, we aim to design a sensing yeast powder based portable test paper, that is, the 'mobile' testing system, take advantage of querom sensing system in bacteria, to achieve the testing of specific microorganism caused disease. In the same time, we built a frame of testing any pathogen that will cause diseases, using different the input and output combination. Furthermore, in order to achieve the simultaneous testing of different pathogens, we design a “fast-shifting box” to accomplish the combination of input and output signaling. This will in theory

Team Tsinghua-A: Synthetic gene switch shows adaptation to DNA copy number variation

In some natural and synthetic biological networks, DNA copy number which transfection into cells is fluctuant-influencing gene expression. We hope target gene expression level has a strong adaptability and ability to DNA copy number by using the method of engineering and bringing in incoherent feed-forward circuit. The robust circuits we designed may apply to cancer detection and gene therapy in the future. Generally speaking, we modeled three and four nodes motifs to find some appropriate circuits, which function reliably in the face of fluctuating stoichiometry of their molecular components. Two designed circuits have been tested and we found that the motifs has certain robustness to DNA copy number.

Team Tsinghua-E: Darwinian evolution for microbial cell factory:in vivo evolution engineering towards tryptophan-overproduction superbug

Darwinian evolution shows great power in creating incredible biological function in amazing speed. Inspired by this, our team aimed at creating novel fast and irrational microbial cell factory by simulating natural Darwinian evolution process. With tryptophan as target product, a novel tryptophan biosensor utilizing translating ribosome mechanism was firstly developed as the foundation for tryptophan productivity and selection pressure switch module. We further constructed this tryptophan overproduction selection gene circuit coupling with in vivo mutation machine (mutator gene of mutD). By fine-tuning the selection conditions, our selection circuit showed good tryptophan dependent growth property, which provides the foundation for further evolution. As a preliminary result of this project, we successfully evolved an ancestor with zero productivity to a high-tryptophan producer only after several rounds of evolution.

Team TzuChiU Formosa: Hypnoseq.

The new pattern of antibiotic resistance is a spreading global issue that may soon leave us defenseless against bacterial infections. Taking a closer look, the lack of comprehensive pharmaceutical management system in Taiwan has come to our concern as it results in easy access to antibiotics. Large amount of antibiotics are added in the forage of animal husbandry and aquaculture,hence, leading to the increase of antibiotic resistance in Taiwan. In order to ameliorate this growing threat, we attempt to carry out “Hypnoseq.” to make this world a better place. Our aim in this project is to combine the sense and antisense mRNA of the antibiotic resistance gene to inhibit the expression of the antibiotic resistance gene. Knowing that they have the ability to conjugate and deliver our designed plasmid to other bacteria, we are able to predict that they can decrease the percentage of antibiotic resistance in the environment.

Team UESTC: Nebula

Nebula is a biological circuit design tool composed of Interactive Part & Automatic Part. We classified the parts released in 2013 and constructed a database for users to choose what they want. In the first part, you are free to link any parts that we have already classified together to meet your requirement. In the second part, once you determine the inducer and the product, our software will offer you the optimized circuit with the input and output that you designated. We use Analytic Hierarchy Process to score every part and edges (passage linking two parts) according to attributions including availability, usefulness, sample status, part status and sequencing. According to weight of edges, we regard the shortest passage between input and output as the optimum presented to users. You can also save the circuits made in Nebula in case you want to check or change it later.

Team UESTC Life: Multistage Degradation of Environment Haloalkanes Contaminant by Co-expression Enzymes

1,2,3-Trichloropropane (TCP) and an organic pesticide-Hexachlorocyclohexane (Lindane-HCH) have been shown to be serious pollutants as they are toxic and quite persistent in the environment, and need to be removed to low levels from polluted sites. Microbial degradation of these compounds represents an important and efficient way to fulfill the target. In order to improve biodegradation efficiency, several powerful genetically engineered E. coli strains have been constructed by the co-expression of key enzymes involving in the biodegradation pathways of the two compounds. For this, foot and mouth disease virus 2A peptide and polycistronic co-expression strategies were adpoted. The results showed that all enzymes could co-expressed as a soluble protein with 2A peptide acting as a linker. Moreover, the resulting engineered E. coli exhibited an excellent capability for the degradation of TCP.

Team UI-Indonesia: Project Blue Ivy - scFv with Blue Indicator as a Biosensor for TB

Tuberculosis (TB) is a worldwide major health problem which infects one third of the world’s population. The absence of reliable diagnostic tool in suburban area, where TB cases are most likely found, is still a great obstacle in TB eradication effort. Seeing Indonesia as one of the high burden countries for TB, UI-Indonesia iGEM team are trying to create a reliable, portable, and easy to use diagnostic tool for detecting TB. We are constructing a biosensor consist of highly specific antibody bound to a fragment of β-Galactosidase as a reporter to detect the presence of protein Ag85, a novel TB biomarker. Our goal is to make a biosensor that will detect the presence of antigen 85 in blood serum of TB suspect. Positive result will be indicated with easy to detect blue color, and when it’s negative, no response will be observed.

Team USTC-Software: Gene Network Analyze and Predict (gNAP)

Synthetic biology creates and uses standardized parts such as Biobricks to build engineered bacteria for various function. To realize those purposes, importing exogenous genes to target bacteria is universal and essential. In this approach, improve or reduce the expression of target genes through interaction is inevitable. Experiments in wet lab could find the effect and choose the best of imported exogenous genes but take a long period of time. gNap utilizes Internet databases to construct a gene regulatory network (GRN) and analyze the effect of exogenous gene by Michaelis-Menten equation and sequence alignment algorithm. Meanwhile, to guide wet lab experimenters to find the best imported gene in the whole network, we use PSO method to figure out the best regulation patterns of new imported genes meeting experimenters’ goal. To realize those ideas, we build gNAP that provides researchers with gene network analysis and prediction.

Team USTC CHINA: T-VACCINE

T-VACCINE is a vaccine initiating immune response by penetrating the skin with the aid of transdermal peptide. From now on, injections are simply history.Based on the theory of user-friendly, a special group of engineering bacteria which produce T-VACCINE is used to create a brand-new 'band-aid' serving as a guardian of our health .We have found a kind of transdermal peptide TD-1,a magical molecule that enhances the permeability of the skin as well as draw filamentous bacteriophages into the skin.By combining the gene fragments of antigen,immune adjuvant LTB and Luman-recruiting factor TNLFα with that of the TD-1, our team got the permeable fusion protein. In order to obtain large amount of extracelluar protein, we chose bacillus subtilis WB800N as our expression chassis. Further more, the universality of our experimental method is verified by the adoption of various antigen of existing vaccine, such as HBsAg, PA and AG85B.

Team UT-Tokyo: Multicellular Analog Clock

We designed a 'multicellular' E.coli clock with a clock hand. Your naked eyes see the red clock hand moving along a circle of E. coli population on an agar plate. The clock hand, expression of mCherry gene, is driven by an “engine” which is constructed under the inspiration of the mechanism of action potential conduction in nerve cells. The engine consists of a positive feedback loop of AHL and negative feedback loops of TetR, AiiA and 2 types of artificial sRNA. We also designed UV reset devices using UV sensor construct. In addition, small RNAs were designed for metabolic engineering of E. coli, which is the first trial in iGEM competition. We show you the new and easy approach in genetic engineering with the BioBrick parts, which will lead to future application of sRNAs in synthetic biology.

Team WHU-China: Master of Regulation: dcas9-based Multi-stage Gene Expression Regulator

Cas9 is an RNA-guided dsDNA nuclease utilized by bacteria immune system. The genetically engineered Cas9 has recently been shown to have the ability to repress or activate desired gene expression. </p>In practical research and industrial application, we usually face the problem to express a gene at different levels, not only “on” or “off ”, so a more flexible regulation method is needed. To achieve multi-stage regulation of target genes, we further develop several dCas9 devices in which dCas9 alone or fused with omega subunit of RNAP is directed by various guide RNAs to different regions of designed double promoters. Therefore, promoters with disparate strength can be either activated or repressed respectively and multi-stage gene expression can be achieved. Also, based on such novel technology platform, we are developing diverse applications such as a guide RNA-mediated oscillator.

Team XMU-China: A SynBio Oscillation Signal Converter

Oscillations permeate every corner of the world, from the alternative current AC in power lines to our tiny microorganism friends. To use oscillations in bacteria as a strong and steady signal transmission method like AC, we need to tackle with the noise of transcription and translation in the cellular environment by coupling millions of cells through the synchronizing genetic oscillations in E.coli. At the colony level cells could be synchronized via quorum sensing, which is limited to tens of micrometers by the AHL, and between colonies a gas-phase redox (mainly H2O2) will serve as a signal that can give positive feedback to the whole circuit over millimeter scales simultaneously. On a liquid crystal display (LCD)-like microfluidic array bacteria grow in separate colonies, so that synchronization in both levels could be verified visually. Now a robust synthetic biology signal converter is accomplished and ready to show the growth environment of cells.

Team XMU Software: Biobrick evaluation and optimization software suit and lab assistant tool

The biobrick evaluation and optimization software tool suit (Brick Worker) provide analysis of biobrick sequences, namely, promoter, RBS, protein coding sequence and terminator. We use PWM algorithm to evaluate the relative strength of promoters and RBS and precisely locate the key region of the sequence that affect its performance. Through codon optimization and GA algorithm our program can analyze and then optimize the protein coding sequence so as to enhance the protein expression level. Terminator efficiency prediction is also included in this suit. As for the lab assistant tool (E’Note), it is a powerful experimental recording platform with exhilarating functions such as multi-line operating, software tool integration and template customization, providing a all-round as well as customized tool to significantly enhance the efficiency of experimental work.

Team ZJU-China: A Tale of Aptamers: Ghost and Elf

This year we aim to utilize aptamer to specifically detect and clear molecules of different sizes. In order to detect and clear certain protein, we make tunneled E.coli called bacterial ghost that allow protein to diffuse in. We then build two types of inner-membrane protein scaffold, which will dimerize when pulled together by two aptamers attached to two sites of the protein. The dimerized proteins have enzymatic activity that can be detected via commercial test strips. The device will also sequester the proteins and allow us to clear them. In order to efficiently detect and clear a small molecule called atrazine, which is an herbicide causing tremendous environmental problems, we split our aptamer-based detection module and clear module into two strains. The first strain is chemotaxic to atrazine and will release quorum sensing molecules to attract the second strain, which contains atrazine hydrolase to clear it.

EUROPE

Team AMU-Poznan: sh-miR designer - tool for construction of RNA interference reagents: sh-miRs

sh-miR Designer will be a software aimed at fast and efficient design of effective RNA interference (RNAi) reagents - sh-miRs, also known as artificial miRNAs. sh-miRs are RNA particles whose structure is based on miRNA precursor pri-miRNA, but sequence interacting with transcript is changed depending on research purpose. Maintenance of structure of pri-miRNA is very important to enable cellular processing and therefore ensure functionality of artificial particles. sh-miRs delivered to cells on genetic vectors - plasmids or viral vectors - enter natural RNAi pathway and silence target mRNA. They can be used in genetic therapies and basic biomedical research.

Team ATOMS-Turkiye: Project Oncoli

According to the World Cancer Research Fund, the estimated number of cancer cases around the world every year is 12.7 million and is expected to increase up to 21 million by the year 2030. Taking this widely popular and alarming obstacle into attention, we have devised a system which is aiming to tackle cancer from a very different perspective to before. Our choice of bacteria Nissle 1917, a probiotic strain of Escherichia Coli, once inside the body will secrete a cancer tracing protein which recognizes and builds up around the cancer cells. Using the quorum sensing system, E.coli Nissle 1917 detects the bacteria inducing substance AI-2 produced by the tracing proteins. Nissle 1917 bacteria motion towards the region of AI-2 and once in the region, produce our cancer killing protein called apoptin. Apoptin enters the cancer cells and induces apoptosis thereby eliminating their existence.

Team Baskent Meds: Killing Legionella pneumophila Softly

Legionella pneumophila is the cause of the Legionnaires' disease which is a type of pneumonia. The bacterium is found in warm water environments, particularly in artificial water supply systems such as air conditioning systems and cooling towers. The enfection occures by inhalation by small droplets of contaminated water. Our aim, as the team “Baskent_Meds”, is developing bacteria which can recognize Legionella pneumophila specifically at species level by legionella quorum sensing, and respond by producing anti-Legionella peptide which is produced by some Staphylococcus strains. Quarum means “minimum”. Legionella pneumophila should sense the minimum amount of cells around to colonize in the environment and express its virulence. So our modified E. coli may sense the presence of Legionella pneumophila in any contaminated surface and kill it.

Team BGU Israel: P.A.S.E. - Programmable Autonomous Self Elimination

Bioremediation and biosensors often require the release of genetically modified organisms (GMOs) to the environment. After being released, these GMOs are no longer under direct control. As their effect on the environment is unknown, they pose a potential threat. In order to eliminate this threat, we are developing a genetic circuit, using e. coli as a model GMO, that limits the lifetime of a bacterial population after it is released to the environment. Our goal is to allow the end user to program a GMO population to survive in the environment until it has completed its task, after which the entire population will disappear without any further external intervention. We employ two approaches to achieve this goal: One relies on the dilution of a synthetic control element through cell division, and the second is based on the lifetime of an essential protein containing an unnatural amino acid.

Team Bielefeld-Germany: Ecolectricity – currently available

There is a growing interest in the use of ecologically friendly alternative energy sources because of the depletion of fossil fuels and an increasing environmental pollution. Therefore, we are developing a Microbial Fuel Cell (MFC). The goal of this project is to generate electricity with a modified Escherichia coli in a self-constructed fuel cell. Besides the technical optimization of the fuel cell, we investigate different genetic approaches like integrating porines and cytochromes as well as endogenous mediators. Using heterologous expression of pore-forming transmembrane proteins, we are able to enhance the extracellular electron transfer, leading to higher membrane permeability. Direct electron transfer can be achieved by integrating cytochromes into the cellular membrane, whereas a production of endogenous mediators enhances the electron transport to the electrode. With different aspects for technical and genetic optimization we enable Ecolectricity, the use of E. coli for direct energy production.

Team Bonn: LOV Wars - May the light be with you

A reliable, yet easily adaptable mechanism for controlling protein activity is key to most areas of life and medical science research. Still, the most common approaches suffer from various flaws. iGEM Bonn 2013 aims to overcome these drawbacks by engineering a novel tool based on blue light-inducible degradation of targeted proteins.</br> The use of a modified ClpXP protease system allows a significant increase in rate and scale of activity change while keeping the modification of the target protein to a minimum. Combining this system with a tool for photo-activatable heterodimerisation based on a LOV domain results in a superior tempero-spatial control.</br> To demonstrate the capabilities of our device, we designed a photosensitive kill-switch. This contributes to the security of synthetic biology in such a way that bacteria accidentally brought out of a safe work environment, for example a red-light-hood, would be killed by sunlight within a short period of time.

Team Bordeaux: The Dairy Planet

The economical stakes of food-processing industry have always been a concern in society. Technological innovations have improved the yield and production costs of daily use products. Advances in health sector and biotechnology made it possible to offer food products rich in substances that are nutritious and possess medicinal properties. Our project aims at producing a new range of lactic cultures able to produce natural flavours and colouring substances in a yogurt; including ones producing resveratrol, a molecule responsible for the red wine beneficial effects, implicated in the 'French paradox”. Necessary routes of biosynthesis will be introduced in Lactobacillus bulgaricus and Streptococcus thermophilus, agents of lactic fermentation. Thus, a work of optimization on the genetical modifications of lactic bacteria has been done. This project will allow an easier production of custom yogurts with beneficial and healing properties, avoiding the use of substances derived from expensive chemical synthesis harmful to the environment.

Team Braunschweig: Engineering synthetic microbial consortia

Bacterial consortia offer a great benefit for synthetic biology due to the ability to perform complex tasks by splitting the whole reaction into smaller reactions and share the task among different specialized strains. Also, a self-regulating bacterial culture with intra consortial dependencies offers great advances in biosafety. To shut down the whole bacterial consortium, only on strain has to be eliminated. We engineer three different E. coli strains to grow in a consortium exploiting different Quorum Sensing systems. Each strain maintains a constitutive expression of an inactive transcription activator (LuxR, LasR or RhlR). Inducers are synthesized by different synthases (LuxI, LasI or RhlI) that are each expressed in one strain and subsequently secreted into the medium. Once taken up by a cell, the inducers bind to the corresponding, inactive transcription factors to render them functional. As a result, an antibiotic resistance under the control of an inducible promoter is expressed.

Team DTU-Denmark: Requiem for a Stream: From Ammonia Pollution to Energy Production via Denitrification

Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from over-fertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. Using two E. coli mutants built with genes from Nitrosomonas europaea and Pseudomonas aeruginosa, we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, and release a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.

Team Dundee: ToxiMop

The ToxiMop project attempts to tackle the problem of freshwater algal blooms by detecting, reducing, and reporting the levels of the algal toxin microcystin. This toxin causes liver damage and is also speculated to be a carcinogen. Microcystin’s toxic action lies in its ability to bind to the human Protein Phosphatase 1 (PP1), which is a major regulator of cell division, protein synthesis and other essential processes. Using synthetic biology techniques, we engineered bacterial chassis (E. coli and B. subtilis) to express PP1, which covalently binds to microcystin. The engineered bacteria can then be used as a molecular mop, the ToxiMop, to remove microcystin from contaminated water. Applying mathematical modelling to our experiments, we optimised our prototype ToxiMop. Additionally, we attempted to develop a biological detector for microcystin, which was combined with our electronic device, the Moptopus. This device has the potential for real-time monitoring and analysis of water bodies.

Team Edinburgh: WastED

The Edinburgh iGEM 2013 team, WastED, is focusing on remediation and valorization of industrial waste streams, with a particular focus on Scottish leather and whisky industry waste waters, containing toxic heavy metal ions as well as fermentable organic components. Using Bacillus subtilis as chassis, we are engineering organisms to capture ions using chelators and metal binding proteins, and to ferment organic components to produce biofuels. We are also testing a new assembly procedure, GenBrick, based on the Genabler assembly system. GenBrick allows assembly of multiple RFC10-compatible BioBricks in a single reaction, and is also well suited to the preparation of fusion proteins and addition of terminal tags. Enzyme fusions may enhance metabolic pathways through substrate channeling. We are testing the effect of protein fusions on fermentation efficiency for biofuel production. In addition, we are examining the implications of possible Scottish independence, following the 2014 referendum, for synthetic biology in Scotland.

Team EPF Lausanne: Taxi.Coli: smart drug delivery

EPF_Lausanne’s team is proud to participate to iGEM 2013 and excited to present their project: Taxi.Coli: smart drug delivery. The team’s vision is to build a biosynthetic drug delivery concept. The key word of this project is “adaptability”. Our goal is to explore a way of using E.Coli as a highly modular carrier, opening the gate to several applications and alternatives in disease treatments. Using the principles of synthetic biology, we engineered a gelatinase secreting E. Coli able to bind gelatin nanoparticles using a biotin-streptavidin interaction and release them in a corresponding location. The drug delivery system is built in three parts: 1) the nanoparticle binding and 2) the environment sensing that 3) triggers the gelatinase release of the engineered E. Coli, liberating the content of the nanoparticle. The nanoparticles made of gelatin are able to carry any type of organic compound leading to a wide range of applications.

Team ETH Zurich: Colisweeper: The world's first biological Minesweeper game

Colisweeper is an interactive, biological version of the Minesweeper computer game, based on luxI/luxR quorum sensing and chromogenic enzymatic reactions. The goal is to clear an agar “minefield” without detonating mines. Genetically engineered Escherichia coli colonies are used as sender-cells (mines) and receiver-cells (non-mines). Mines secrete the signaling molecule N-(3-oxohexanoyl)-l-homoserine lactone (OHHL) whereas non-mines process the signal. To distinguish between OHHL-levels, a library of PLuxR promoters with various sensitivities was created through site-saturation mutagenesis. High-pass filters were constructed to control the expression of different orthogonal hydrolases in non-mines, depending on the number of surrounding mines. Additionally, the mines express their own hydrolase. A spatiotemporal reaction-diffusion model was established to evaluate and improve the system. To play Colisweeper, a colorless substrate solution is pipetted onto a colony of choice. The result is a defined color change within minutes, allowing identification of the played colony and the number of mines surrounding it.

Team Evry: Iron coli Project

This year, our project focuses on diseases that are subsequent to an iron overload such as hemochromatosis and thalassemia. </p> Nowadays, iron overload is mainly treated by bloodlettings for hemochromatosic patients but this treatment cannot be extended to thalassemic patients who suffer from anaemia. The aim of our project is to prevent the intestinal absorption of iron by engineering Escherichia coli to produce siderophores, chelators of iron. This strategy acts directly at the source. </p> We engineer Escherichia coli using the Ferric Uptake Regulation (FUR) couple to an inverter system, in order to produce these siderophores in presence of iron. To reduce the patient's iron absorption, our bacteria is encapsulated in a pill. Once it arrives in the duodenum, our bacteria will produce the siderophore at their full potential and chelate the iron. </p>

Team Exeter: Paint by coli: Creating a Colour Bio-camera Using Escherichia coli via complete optical control

Synthetic biology has lead to microorganisms being pushed into an unprecedented range of novel functions. Many bacterial systems currently rely on external stimuli to induce transcription. One dimensional protocols often require constant monitoring of applied chemical concentrations, leading to them becoming inept for more complex systems. A triplet of NOT gated photoreceptors in Escherichia coli, will be used to create a system which is finely controlled using only light. This will be showcased using magenta, cyan and yellow pigments as outputs. Varying the intensity and wavelength of light projected onto E. coli will control the shade and colour produced, respectively. Hence, this will show the versatility of the optical control by creating a full colour bio-camera. Additionally, using bacteria to produce an image vastly increases the resolution when compared to conventional cameras, due to the micrometre scale of bacteria.

Team Frankfurt: Steviomyces - sweeter than sugar

The Stevia plant produces several sweeteners known as Steviolglycosides which have only recently been admitted as a foodadditive in the European Union. The iGEM-Team Frankfurt 2013 searches for ways to transfer the pathway of the plant into Saccharomyces cerevisiae in order to make stevia production possible with both lower effort and lower costs. Several of known problems with carbohydratesweeteners like diabetes or caries could be overcome by the Steviolglycosides which are produced by Stevia rebaudiana. We're building upon results gained from last year's competition which gave us the possibility to transfer a mevalonat plasmid into yeast to increase the production of a steviol-precursor Geranylgeranyl-diphosphate. This year we're searching for a further reconstruction of the pathway and transfering the 2nd plasmid for synthesis of Steviol from Geranylgeranyl-diphosphate into yeast. Thus the whole pathway can take place in a microbial organism and easify the production by lowering the costs.

Team Freiburg: uniCAS - The Toolkit for Gene Regulation

Our Team developed a universal toolkit, termed uniCAS, that enables customizable gene regulation in mammalian cells. Therefore, we engineered the recently discovered and highly promising CRISPR/CAS9 system. The regulation is based on the RNA-guided CAS9 protein, which allows targeting of specific DNA sequences. Our toolkit comprises not only a standardized CAS9 protein, but also different effector domains for efficient gene activation or repression. We further engineered a modular RNA plasmid for easy implementation of RNA guide sequences. As an additional feature, we established an innovative screening method for assessing the functionality of our uniCAS fusion proteins. Single genes and even whole genetic networks can be modified using our uniCAS toolkit. We think that our toolbox of standardized parts of the CRISPR/CAS9 system offers broad application in research fields such as tissue engineering, stem cell reprogramming and fundamental research.

Team Gdansk-UG: MetOli

The aim of our project was to construct a biological system that would be able to detect methanol in ethanol solutions. Our idea was to create a test that could be performed not only in the laboratory, but also at home. We believe that such test would reduce the rate of intoxications by methanol during ethanol consumption. To achieve it, we used a methanol-dependent promoter from Methylobacterium organophilum which would control the production of a dye, for instance GFP, or an enzyme that would produce visible product, such as catechol oxidase. Our eventual goal is to find a bacterium that would not only react to methanol, but also survive in high concentrations of ethanol.

Team Goettingen: The beast and its Achilles heel: A novel target to fight multi-resistant bacteria

Since the discovery of penicillin by Alexander Fleming in 1928, antibiotics have marked a major victory of mankind in the battle against infectious diseases. However, after 90 years, the antibiotics are now losing their old time glory: Bacteria acquire resistance against antibiotics and become unbridled. We must control the use of antibiotics, meanwhile, we need new antibiotics, which can suffiently eliminate the invaders without hurting the 'good' bacteria. Therefore, c-di-AMP, an important, recently discovered signaling molecule in gram-positive bacteria, has come to our sight. Our project is to build a screening system targeting c-di-AMP, which could be applied in novel-drug screening. With this system, the level of c-di-AMP in the cell can be visualised and measured.

Team Grenoble-EMSE-LSU: Light Automated Cell Control by Talk’E. coli

Maintaining cell growth state during culturing is generally difficult due to metabolic adaptation and changing cell division rates. Using light-induced promoters and a phototoxic fluorescent protein, we've designed Talk’E. coli. It uses light signals to communicate with bacteria allowing the researcher to remotely control the cultures using a computer. Cell density is monitored through fluorescence recordings and, thanks to a predictive model, Talk’E. coli responds by illuminating the culture with one or more wavelengths to obtain different effects: killing off cells beyond a threshold density, or producing or degrading protein. The tool is portable and mountable in an incubator making it a handy device for research.

Team Groningen: Engineering Bacillus subtilis to self-assemble into a biofilm that coats medical implants with spider silk.

Approximately half of all implanted medical devices result in one or more medical complication, which have been found to increase mortality rates by 25%, and to cost the amerian society an additional 30 billion dollars every year. A possible solution for these complications is to form a protective biocompatible layer between the implant and the body by means of a spider silk coating. This is achieved through mathematical modelling, techniques from the synthetic biology, and the Gram-positive bacteria Bacillus subtilis, which is redesigned to secrete silk and to self-assemble into a biofilm surrounding the implant. It uses a modified chemotaxis system coupled to the DesK heat sensing system to do so. B. subtilis is furthermore often used in the industry for the commercial production of extracellular proteins, and is generally regarded as safe.

Team Heidelberg: THE PHILOSOPHER’S STONE

Several secondary metabolites, such as commonly used antibiotics, pigments and detoxifying enzymes, are synthesized by non-ribosomal peptide synthetases (NRPSs). These enzymes beautifully reflect one of the fundamental principles of synthetic biology, as they are remarkably modular. We will assemble new NRPSs by combining individual domains and modules of different origin, thus setting the basis for novel and customized synthesis of non-ribosomal peptides. To make the use of NRPSs amenable to a wider community, we will devise a new software-tool, called “NRPS Designer”, which predicts the optimal modular composition of synthetic NRPSs for production of any desired peptide and outputs a cloning strategy based on Gibson assembly. As an application relevant to society, we will engineer Escherichia coli to recycle gold from electronic waste in a cost- and energy-efficient way through the heterologous expression of the NRPS pathway of Delftia acidovorans that naturally enables precipitation of gold ions from solution.

Team Imperial College: Plasticity: Engineering microbes to make environmentally friendly plastics from non-recyclable waste

Accumulation of waste represents a considerable problem to humanity. Over the next 50 years, the global community will produce approximately 2 trillion tonnes of waste, or 2.5 times the weight of Mount Everest. Traditionally, mixed non-recyclable waste is sent to landfill or for incineration, both of which result in environmental damage. The detrimental effects are perpetrated by the plastic degradation into toxic byproducts and the production of greenhouse gases by these processes. As an alternative we propose to upcycle this mixed waste into the bioplastic poly-3-hydroxybutyrate (P3HB) to create a closed loop recycling system. Our engineered E. coli will operate within sealed bioreactors. In the future we picture the use of our system in a variety of contexts as part of our M.A.P.L.E. (Modular And Plastic Looping E.coli) system.

Team INSA Toulouse: E. calculus Project

The E. calculus project consists constructing a full n-bits adder capable of transmitting a carry to the next step. The designed strain contains specific devices that should ensure a relatively precise calculation and will be decomposed as follows:
- Various logic gates using specially designed recombinases and recombination sites to avoid reversibility of the gates states.
- A strict control of the expression of recombinases via a tight riboregulation control of the translation of recombinases genes
- A general inducer, switching the strain from inactive to active counting.
- A carry system based on the diffusion of a messenger molecule to the second bit.
- An artificial input system based on photoreceptors sensible to blue and red lights.
The envisioned system should approach as much possible the reliability of an electronic two digit device and may help the Synthetic Biology community designing strong and robust Genetic Boolean Operators.

Team ITU MOBGAM Turkey: Intrinsic Factor-y

Pernicious anemia is described first by James S. Combe in 1822. Pernicious anemia is a type of anemia occurs due to malabsorption of vitamin B12 in the small intestine due to problems with the production of Intrinsic Factor, which is responsible for the absorption of vitamine B12. Pernicious anemia shows its stiking effects on blood, gastro-intestinal tract and nervous system and pernicious anemia usually develops together with an autoimmune disease. Our aim as ITU MOBGAM IGEM Team, is to design a bacterium that is capable of surviving in small intestine and secreting Intrinsic Factor dependent on pH. Also, we design a genetic circuit for controlling the overgrowth and containment of bacteria.

Team Kent: No to NO: A novel approach to reduce greenhouse gas

In today’s rapidly changing environment greenhouse gases such as NO are an issue that need to be addressed. NO has been proven to have a detrimental impact on the environment and iGem Team Kent 2013 will provide a solution that focuses on reducing the amount of NO formed in waste water. Our system will utilise an engineered strain of E. coli which will be capable of converting this excess NO into ammonia. Our Biobricks have been designed to enable the detection of NO using the norV promoter. The NO can then be converted into ammonia via the nitrite reductase enzyme encoded by the E. coli gene NrfA. Our solution will have many advantages over the current approaches to waste water treatment such as reduced cost and risk of contamination. Our system will provide a source of recycled ammonia and could be a greener alternative to the Haber Bosch process.

Team KU Leuven: E. coligy: Plants with BanAphids

Aphids, the little green plant-sucking bugs, can pose serious threats to a farmer's proceeds. Not only physical damage to the crops caused by the sucking is a problem, but aphids also transmit harmful viruses to the plants. The magnitude of loss is difficult to quantify as it changes with aphid species, crop species, location, year and other factors. The use of insecticides to control aphid population is contested, as it has a negative effect on the natural predators and aphids grow resistant. That's why we, the KU Leuven iGEM 2013 team, decided to do something about it in a sustainable way, using an insecticide-free controlling mechanism. With E.coligy: Plants with BanAphids we will teach E.coli cells to hack into insects signaling systems to drive off the aphids and attract the natural predators, such as the ladybug.

Team Leeds: The Micro-beagle - A living biosensor

Micro-Beagle is a novel reporter system for E-coli that, as an iGEM first, has been designed to dynamically detect arbitrary target solids (including other cells) through a mechanism activated by cell surface binding. Micro-Beagle is a modular system, utilising Ice Nucleation Protein to express and position target-binding peptides on the cell surface. Target binding induces membrane stress that activates the Cpx signalling pathway, and Micro-Beagle thus utilises a promoter from this pathway (pCpxR) to initiate expression of a reporter protein, such as GFP. As a proof of concept, we have used silica beads as a model diagnostic target (a pathogen surrogate) and the silica-binding “Si4” sequence as the target-binding peptide. We foresee Micro-Beagle being adapted for both the detection of waterborne pathogens and a variety of other diagnostic applications, and we envision future multisensor Micro-Beagles in which diverse pathogens can be simultaneously and quantitatively measured from a single water sample.

Team Leicester: Biological routes to recycling, re-using and re-purposing polystyrene

Polystyrene is a useful material, but also a visible pollutant that locks up oil-derived hydrocarbons. For 2013 we are diversifying, to reduce polystyrene’s various environmental impacts: Recycling - Building on 2012’s project, we are adapting the toluene degradation pathway from Pseudomonas species to work on polystyrene, in E. coli.</p> Re-using - Consumer 3D printers use a variety of thermoplastics but virgin plastic is usually required. Recycled polystyrene can be a support for making complex 3D shapes, and removed later. Polystyrene is soluble in limonene (an environmentally friendly solvent) so we are adapting limonene biosynthesis biobricks, to enable biological 'finishing' of 3D printed objects.</p> Re-purposing - Polystyrene is a great building insulator, but needs to be flame retardant. Currently this involves adding halogenated hydrocarbons, proven environmental pollutants. Recently DNA was shown to be an effective flame retardant, so we are using synthetic biology to generate cheap DNA, for flame retardant polystyrene.</p>

Team Linkoping Sweden: A novel immunochemical detection system for food allergens.

Antibodies are usefull for recognition of antigens in food. Antibodies have, however, a very complex structure that is not suitable for expression in E. Coli. The Camelid antibody IgG (cIgG), however, has lower complexity than the Human IgG. We present a new approach for recognition of food allergens with a synthetizised cIgG for expression in E. Coli. The epitope of cIgG is designed for Hen Egg White Lysosyme (HEWL). The cIgG is designed with a linker that connects to the bioluminescent enzyme Luciferase. We also synthetizised an HEWL antigen carrying the protein RFP, A-HRFP, that reacts to the luminescence of luciferase as the A-HRFP attaches to the cIgG. The recognition of HEWL in a sample leads to the release of luminescent green-light as a result of HEWL binding to the cIgG. If, however, no HEWL antigen is present in the sample, A-HRFP binds to cIgG resulting in a luminescent red-shift.

Team Manchester: E. c(oil)i; The Lean, Green, Fat-Producing SynBio Machine

From food products, to cosmetics and biodiesel, palm oil is the world’s most widely used vegetable oil. Its demand is ever increasing; however the current method of extracting palm oil is severely unsustainable. Massive deforestation is required to build oil palm plantations, ruining the land of locals in Malaysia and Indonesia. Manchester iGEM aims to combat this by providing a more eco-friendly source of the four main components of palm oil. We reengineered the fatty acid biosynthesis pathway of E. coli to overproduce palmitic and stearic acid and introduced two new genes, desaturase and desaturase, to yield oleic and linoleic acid. To explore the scale-up potential of synthetic palm oil production in E. coli, we developed a fully parameterised kinetic model of the engineered fatty acid biosynthesis pathway.

Team Marburg: Phaectory

The diatom Phaeodactylum tricornutum is a widely spread organism in marine waters. It belongs to the group of diatoms. As a group of great ecological relevance diatoms are responsible for up to 20% of the global CO2 fixation and generate about 40 % of the marine biomass of primary producers. In addition, diatoms represent an important source of lipids and silicate making them interesting for various biotechnological applications e.g. in biofuel industry, food industry and nanofabrication. Furthermore, a relatively easy biolistic method for transfection is established. A simple cultivation eases a putative industrial use of the diatom. Former researches not only proved a possible expression of antibodies, bioplastic and other recombinant proteins, but also demonstrated a direct secretion of the expressed proteins in the outer medium, making it easier to filter the wanted proteins. These characteristics make P. tricornutum an interesting organism for putative industrial use.

Team METU Turkey: Bee subtilis

Taking a major role in pollination, bees are one of the most important organisms within an ecosystem. However their populations are in serious decline. Colony Collapse Disorder has been found as the most common cause of the disappearance of bees in large numbers. In this study, we aimed decrease the number of hives affected by chemical compounds such as imidacloprid.Our plan is to turn the mutualistic bacteria living in bees' guts into a shield mechanism to protect the bees against these factors. A protein CYP6G1 found in Drosophilia melanogaster has the ability to degrade imidacloprid into harmless substances. Moreover, coumaric acid increases the general immunity of bees against harmful components and we aim to increase the level of coumaric acid in bees' guts. The main objective of this study is the transformation of the genes coding for these two proteins to Bacillus subtilis, which mutualistically live in bees' guts.

Team Newcastle: L-forms: Bacteria without a cell wall - a novel chassis for synthetic biology

L-forms are bacterial without cell walls that are still able to divide without the normally essential cell division machinery. The lack of a cell wall imparts a range of interesting properties and we show that L-forms can be used as a novel chassis for a range of fundamental applications in synthetic biology. We produced a BioBrick for Bacillus subtilis, that allows cell morphology to be toggled from normal to L-form. We have explored some of the interesting opportunities that L-forms provide including cell fusion, genome shuffling and the generation of differently shaped cells using microfluidics. L-forms are thought to exist naturally within plant tissues and we also studied their use as agents for delivering novel functionality into plants. For project outreach, we created a game as an Android application and considered the implications raised by our project and also look at the exciting relationship between synthetic biology and architecture.

Team NRP-UEA-Norwich: Developing Biosensors to Identify Antimycin-Producing Actinomycetes

Antimycins, anti-fungal compounds primarily produced by Streptomyces (a sub-set of actinomycetes), function by inhibiting the final stage of the electron transport chain. Our aim is to develop Biosensors to aid identification of novel antimycin-producing actinomycetes. Homologues of the AntA sigma factor, the key regulatory protein in antimycin biosynthesis, are present in all 14 known biosynthetic gene clusters. Due to this property, Biosensors have been designed with the AntA-regulated promoter (antGp) controlling the expression of three reporters: neomycin resistance gene, RFP (red fluorescent protein) and GUS (providing β-glucoronidase activity). The Biosensors will be produced, trialled and optimised where possible after sub-cloning into two actinomycete-specific integrative plasmids, pMS82 (BT1 integrase) and pAU3-45 (C31 integrase). Worldwide soil and sediment samples have been collected to produce a library of actinomycete strains, which will be screened using our Biosensors, the ultimate goal being to screen bacterial strains for antimycin production.

Team NTNU-Trondheim: VesiColi

Gram negative bacteria produce outer membrane vesicles (OMV) in the size range of 20-200nm. Whereas their function and contents has been studied for decades, their potential as drug carriers has not been investigated before. We want to introduce protein G from Streptococcus dysgalactiae subsp. equisimilis into Escherichia coli OMV's. Protein G is known to bind to human serum albumin (HSA) which helps S. dysgalactiae subsp. equisimilis hide from the immune system.

The second part of our project is to introduce fluorescent proteins (FP's) linked together into the vesicles. Introducing protein G and linked FP's into the vesicles will demonstrate that it is indeed possible to manipulate the content, and therefore the properties, of OMV's.

Team Paris Bettencourt: Fight Tuberculosis with Modern Weapons!

We are testing new weapons for the global war against Mycobacterium tuberculosis (MTb), a pathogen that infects nearly 2 billion people. Our 4 synergistic projects aim to help in the prevention, diagnosis, and treatment of tuberculosis. 1) We are reproducing an essential MTb metabolic pathway in E. coli, where it can be easily and safely targeted in a drug screen. 2) We are building a phage-based biosensor to allow the rapid diagnosis specifically drug-resistant MTb strains. 3) We are constructing a mycobacteriophage to detect and counterselect drug-resistant Mtb in the environment. 4) We are programming E. coli to follow MTb into human macrophages and saturate it with bacteriolytic enzymes. We want to vanquish tuberculosis and build a TB-free world.

Team Paris Saclay: PCBbusters

PCBs (Polychlorobiphenyls) are synthetic chemicals widely used during the late 20th century. These compounds are extraordinarily stable, not readily biodegradable and have accumulated in the environment. PCBs also accumulate in animal fatty tissues including human tissues. As PCBs are probably carcinogenic and some are endocrine disruptors, they constitute an important health issue. Although PCBs have no natural equivalents, some bacterial communities have developed the capacity to degrade PCBs. Highly chlorinated PCBs undergo anaerobic reductive dechlorination, lowering the chlorine atom number. Lightly chlorinated PCBs are then degraded via the aerobic biphenyl degradation pathway. Our project is to construct an Escherichia coli strain capable of degrading PCBs by introducing in the strain genes involved in PCB degradation in various bacteria. Because some steps are anaerobic and others aerobic, we want to use an oxygen-based regulation of gene expression. We also want to develop a sensor system to detect PCBs in the environment.

Team Poznan-BioInf: SR-MUX: a biological multiplexer with 3-bit editable transcriptional memory.

Our goal is to engineer a device allowing to save up to three binary input signals in living E. coli cells, resulting in expression of red, blue and green fluorescent proteins as reporters. Converting inducer signals into expression of serine recombinases, enzymes capable of specific DNA editing, we are able to create three transcriptional analogues of transistors - transcriptors - and to use them as elemental memory units called SR-latches under control of a fourth, strobe signal, providing a mean to reset the system to its original state. This complex biological memory unit opens the way to cheap, reversible gene induction, useful both to the industry and researchers, not only lowering inducing cost but also being less stressful for the studied organisms, e.g. plants. It is also another step towards Von Neumann-inspired biocomputers.

Team SDU-Denmark: Bacteriorganic Rubber

The growing demand for natural rubber causes deforestation of the rainforest or occupation of arable lands, all due to the founding of new plantations. If producing rubber by bacteria succeeds, production of natural rubber will not be limited to the regions where the rubber tree can grow. Our project aims to make an E. coli strain able to produce natural rubber while grown under controlled conditions. Natural rubber is composed of polymerized IPP (isopentenyl pyrophoshate) units. E. coli already possesses the ability to produce IPP, but it lacks the polymerization enzyme, prenyltransferase, from the rubber tree. In this project we introduce prenyltransferase into E. coli and simultaneously manipulate the bacteria to produce more of the IPP links, consequently leading to the production of natural rubber in the bacterial setting.

Team TU-Delft: Peptidor: Detection and killing of resistant S. aureus using antimicrobial peptides

Methicillin-Resistant Staphylococcus aureus causes major problems, especially in hospitals, leading to over half a million infections annually in the US alone. Of the alternative treatments currently under investigation one of the more promising is through antimicrobial peptides (AMPs). These small, highly-specific peptides attack the membrane of target organisms. Thousands of AMPs are known to exist and little resistance against them has been developed. The Peptidor project consists of an E. coli that can detect S. aureus, using S. aureus’ native quorum sensing system, in order to locally produce and deliver AMPs. Upon detection, peptides inactivated by a SUMO-tag fusion, are overexpressed. After a delay period, introduced through a negative transcriptional cascade, a SUMO protease is expressed cleaving off the inactivating tag. Using this mechanism, high concentrations of peptide are delivered at the infection to efficiently kill S. aureus. As a safety mechanism, the timer also activates an E. coli kill-switch.

Team TU-Eindhoven: MRiGEM: Creating a production and delivery system for a CEST MRI contrast agent

Our project presents an alternative solution to the use of heavy metals MRI contrast agents by focusing on CEST MRI. Within CEST imaging, proteins enclosing hydrogen atoms generate high quality images. We use Escherichia coli to create CEST proteins when the bacteria sense a hypoxic environment due to a promoter designed for this purpose, thus working as a production and delivery system for the CEST MRI contrast agent. Hypoxic regions are related to tumors, therefore our eventual goal is to use this device to target and image tumors in humans by injecting the bacteria into the bloodstream. A second application is tracking bacteria in bacterial infections studies. For the iGEM competition however, the proteins are only expressed ex-vivo: in aerobic and anaerobic conditions. We aim to achieve an efficient testing of the CEST properties of the proteins and confirm the promoter’s ability to express each protein.

Team TU-Munich: PhyscoFilter – Clean different

The contamination of aquatic ecosystems with multiple anthropogenic pollutants has become a problem since the industrial revolution. Antibiotics, hormones and various noxious substances threaten environmental health and are not effectively removed by conventional waste water treatment. We propose to employ transgenic plants which produce effectors for enzymatic degradation (BioDegradation) or specific binding (BioAccumulation) of pollutants. The autotrophic, sedentary, aquatic nature of the moss Physcomitrella patens makes it an ideal chassis for a self-renewing, low-maintenance and cheap water filter. A light-triggered kill switch prevents unintended environmental spreading by limiting viability to places where the spectrum of sun light is appropriately filtered. Furthermore, we have developed a device to implement this biological filter in an aquatic environment, investigated the application of this new technology and examined its economic feasibility. Based on our results, PhyscoFilter may become a game-changing approach to improve global water quality in an affordable and sustainable fashion.

Team Tuebingen: Tuebingen Yeast Based Progestin Measurement System

Detrimental alterations caused to water bodies by endocrine disruptive chemicals are an increasing problem in our environment. Especially steroid hormones influence the development and generative behavior of fish. The binding of those hormones to progestogen receptors can mistime the reproductive behavior of aquatic organisms and thereby endanger population balance. Our aim is to construct a yeast-based measurement system for progestin concentration in water samples. Many currently used methods are either very expensive or significantly slower than our method will be. We take advantage of membrane bound receptors in order to achieve high specificity and to speed up measurement. The binding of the ligand to the receptor stops inhibition of the reporter and thereby initiates its expression through a sensitive signaling-chain. This transcriptional switch allows measurement of very small amounts of substrate. To improve our system we use different interchangeable parts for assembly to get a high variety of possible applications.

Team TU Darmstadt: Hunting Fungi

The danger of fungal contamination of grains and cereals but also other food sources has severe consequences. Undetected contaminations can render large quantities of food stocks useless – with detrimental effects on the economy and the food supply. We want to develop a handy device which allows an easy, fast and reliable detection of mycotoxins. For that our team uses various methods from the fields of synthetic biology, electrical engineering and information processing. Our system relies on E. coli with modified TAR receptor interacting with specific mycotoxins. If these are present in the sample they induce a conformational change of TAR and thereby generates a measurable FRET-beacon by bringing two fluorophores in close distance to each other. The modified E. coli will be embedded in exchangeable capsules. Together with a handheld-device and a controlling Smartphone App they will guarantee that measurements can be done quickly, easy to operate and secure.

Team UCL: Spotless Mind

This year, the UCL iGEM team is taking a radical new step with synthetic biology. We intend to explore the potential application genetic engineering techniques on the brain, by tackling Alzheimer's disease, which is linked to the presence of amyloid plaques in the brain. Targets for the project include: establishing microglia cells as a new Synthetic Biology chassis and constructing new BioBricks to enable engineered Microglia to detect and destroy disease-associated amyloid plaques.

Team UCL PG: Spectra

Spectra aim to use a novel configuration of synthetic gene networks (SGNs) to drive evolution of a fluorescent protein with dramatically improved spectroscopic properties. In future we intend to use the capabilities this enhanced fluorescent protein will provide to enable better dissection of differentiation pathways in stem cells.

Team UGent: A new model for chromosomal evolution: Eliminating antibiotic resistance

The main goal of industrial biotechnology is to increase the yield of biochemical products using microorganisms as production hosts. This includes engineering large synthetic pathways and improving their expression. Overexpression of genes has hitherto mainly been achieved by using high or medium copy plasmids. However, studies have demonstrated that plasmid-bearing cells lose their productivity fairly quickly as a result of genetic instability. Therefore a new method was developed for the overexpression of a gene of interest in the bacterial chromosome: Chemically Inducible Chromosomal evolution (CIChE). In this technique the chromosome is evolved to contain a higher number of gene copies by adding a chemical inducer. The original model for CIChE, however, results in bacterial strains containing a large number of antibiotic resistance genes. To make this valuable technique more widely applicable in the industry, we developed a model for chromosomal evolution based on a toxin-antitoxin system instead of antibiotic resistance.

Team UNIK Copenhagen: Project Magneto

Project Magneto is a biological system that allows us to find better ways to treat cancer, acts as a sustainable energy source or just enables us to visualize our environment in a new way. We created it using magnetosomes. Thanks to these specialized organelles magnetotactic bacteria are able to navigate in the earth’s magnetic field. The magnetosome is a nanomagnet which consists of a magnetic crystal housed inside a lipid membrane. Magnetosomes arrange together in chains and act as a compass needle thereby orienting the cell. They show various properties that give them an advantage over industrially synthesized nanomagnets. We demonstrate their usability by fusing fluorescent proteins to their membrane. Through this we open the way for using magnetosomes in various different applications where the fluorescent protein could be simply replaced by a drug for targeted cancer therapy, an ATP-synthase to create a biological dynamo or dye for magnetic paint.

Team UniSalento Lecce: NICKBUSTERS: developing a nickel detection and remediation platform

Nickel is one of the most widespread heavy metals in the ecosystem and, though essential, its excess could be toxic, leading to various noxious effects; nowadays bacteria-mediated bioremediation from inorganic substances seems to be a considerably relevant frontier in microbic biotechnologies. Our project aims to develop a living system in two easy monitorable bacterial platforms who would work as a Nickel detector and a Nickel remediation system. The devices are based on genetic parts from Helicobacter pylori: from the nickel sensing device, H.pylori NikR protein, to the Nickel storage system, Hpn protein, whose role is to store the Nickel ions inside the cell. The two devices are split in two separate populations, which intercommunicate through Quorum Sensing. The system allows to remove the Nickel ions from polluted environmental substrates through bioaccumulation and could be easily implemented in purification plants.

Team UNITN-Trento: B. fruity

B. fruity envisions an environmentally friendly way to control fruit ripening by exploiting an engineered, light regulated strain of B. subtilis. The system works by synthesising ethylene or methyl salicylate (MeSA) upon photoinduction. Everything is housed in a vending machine-like enclosure that regulates fruit ripening in response to consumer demand. Ethylene is a natural plant hormone that is widely used to ripen fruit, such as bananas and kiwi. However, the synthesis, handling, and storage of ethylene is expensive and dangerous. In contrast, B. fruity produces ethylene from inexpensive material by exploiting a TCA cycle intermediate, 2-oxoglutarate, and the activity of P. syringae 2-oxoglutarate decarboxylase. The inhibition of fruit ripening results from the synthesis of MeSA via a pathway built with wintergreen parts. As a proof of concept, we engineered E. coli with the above systems plus the YF1/FixJ blue light receptor device.

Team Uppsala: LactoNutritious

Malnutrition is today a major global problem that affects people both in affluent and developing countries. Even if you get the right amount of calories, if these do not contain sufficient amounts of micronutrients, like vitamins and minerals, serious illness and even death can be the result. The goal of our project is to alleviate this problem by applying synthetic biology to probiotic bacteria. With our project, we will make the lactobacillus genus the new probiotic platform for metabolic engineering of nutritional compounds. We will engineer probiotics to produce for example beta-carotene, resveratrol, p-coumaric acid, miraculin and saffron. To exemplify what this combination of probiotics and metabolic engineering can accomplish we used our modified bacteria to create nutritionally enriched yoghurt. We have also put great effort into addressing the ethical and safety issues that naturally follow when creating GM food.

Team Valencia-CIPF: Project - Freshellent Yeast

Our team will try to develop a project based on the production of aromas and repellents. The aim is to create a biological platform within a model organism, such as common yeast, to develop an alternative method for production of several aromatic monoterpenoids. The advantage of this organism as producer lies in its capabilities of genetic modification, robustness and culture simplicity. We can also control the production of these compounds using different promoters, so we can choose our favourite aroma while there is repellent activity. The microorganism is completely harmless as it is responsible for fermenting bread and beer. The project aims to establish the basis for future production of repellents in a sustainable and organical manner in developing homes that are under the risk of pandemics caused by mosquitoes and other insects.

Team Valencia Biocampus: Wormboys

Bacteria are essential in biotechnology, but they can hardly move. Nematodes, such a C. elegans, are fast crawling organisms, but they have limited biotechnological applications. By combining the best from both organisms, we present the first artificial synthetic symbiosis with bacteria engineered to ride on worms, which concentrate in hotspots where bacteria perform a desired biotechnological process, such as bioplastic (PHA) production. We have engineered Pseudomas putida with a whole operon that allows the formation of a biofilm on the worm. Biofilm formation is swhitched on and off depending on the media, and thus bacteria get on and off the worm like travellers on a bus. We have also engineered a third partner, E. coli, to express an interference RNA that promotes clumping. Taken together, our artificial symbiosis allows biotechnologically interesting bacteria to travel on nematodes, reach nutrient-rich biomass spots and maximize the efficiency of biotechnological fermentations in heterogenous substrates.

Team Wageningen UR: Aspergillus niGEM: A lov story

The fact that secondary metabolites are often synthesized as polymer backbones that are subsequently diversified greatly via the actions of tailoring enzymes sets the stage for combinatorial biochemistry because their biosynthesis is modular. One of the goals is to establish a modular system of domain shuffling to generate a plethora of novel enzymes with new and improved functionalities. The production of lovastatin, a drug used in lowering LDL cholesterol for patients suffering from cardiovascular disease, has been chosen as a proof of principle. The aim is to transfer the entire lovastatin metabolic pathway from A. terreus into a GRAS organism like Aspergillus niger. To expand our scope we will also be working on host engineering, trying to create a single cell phenotype of Aspergillus niger. To increase the accessibility of our host we also deliver a set of tools, which include ATP and pH biosensors, cytoskeletal gfp-fusions and chromoproteins.

Team Warsaw: FluoSafe

We are presenting to you FluoSafe- a biosensor for acrylamide, known for its carcinogenic and neurotoxic effect! This compound is present not only in biological laboratories but also in starch-based food products (fries, chips etc.). We aim to construct a bacterial strain that would serve as a detector of acrylic amide. This will be attempted in two ways: through the use of roGFP (redox sensitive GFP) fused with glutaredoxin 1 (the presence of acrylamide is known to affect the cellular gluthatione pool) and by expressing hemoglobin α- and β- subunits fused with split fluorophore (adducts formed by acrylamide on the N-terminal valine are known to affect interactions between subunits). We also constructed a BiFC toolbox in BioBrick standard. We sought to find out what was the effect of acrylamide on a variety of human cell lines and asses the toxicity of different concentrations of this compound.

Team Westminster: Hungry for chitin

This year the Westminster iGEM team are tackling the growing bed bug problem. Serratia marcescens has been identified as an efficient chitin degrader, however as it is a pathogenic organism it can not be used as a biocontrol agent. Our idea is to use the chitin genes from this bacterium and create a chitin degrading E.coli. We will test the efficiency of the activity of chitinase which is expressed by our engineered E.coli compared to that of S. marcescens by using a chitin azure assay.

Team York UK: Electricus Aureus: Our greatest source of power comes from the smallest organisms on Earth

We envisage a world where your mobile phone my one day be powered by synthetically engineered microorganisms, when non-renewable energy is a thing of the past. Our project comes at a time when all sources of energy are fighting to be the lesser of many evils; we would therefore like to propose a cheaper, greener and more effective source of energy. Currently, fuel cells do not produce sufficient power to be used for household appliances. Our genetically engineered organism will help us change this and be the first step in the Renewable Revolution. Bacteria are the most abundant form of life on Earth, they survive in harsh environments and they divide rapidly. Thus, they can be a renewable, sustainable source of energy. Our organism will deposit gold nanoparticles on the battery to increase its conductivity. These gold ions come from toxic pharmaceutical waste which is extremely harmful to the environment.

LATIN AMERICA

Team BIOSINT Mexico: Smartpro

This year Biosint Mexico team will be developed a smart probiotic. Along the competition have been present several projects about probiotics, nevertheless the main disadvantage was that most of them were not being created in a lactobacillus strain. Because of this we constructed a Lactobacillus platform for others iGEM teams. Continuing with the idea of the smart probiotic system we include a sensor for xenobiotic substances that could detect and decrease intoxications by pesticides. Also the team implemented a kill switch for safety issues. This project contributes to resolve one of the Mexican food and health problems.

Team Buenos Aires: To drink or not to drink

Our project is focused on developing a biosensor specific for certain water pollutants, with a modular and scalable approach. This approach would make it easy to adapt the response for the detection of different substances. In contrast to other iGEM biosensors, it does not rely on expensive equipment or qualified people to interpret the results. Being aware that most of the populations affected by consumption of contaminated groundwater don’t have scientific or technical training, we intend the device to be cheap and easily distributed. We have designed it in a way that any user could easily determine the presence and level of the contaminant on drinking water, using image-based instructions. The project will focus on measuring a primary pollutant: arsenic. However, its modular and scalable design provides an easy way to measure various contaminants such as nitrate/nitrite among others.

Team Ciencias-UNAM: Skully coli

The human peptide LL-37 is an antimicrobial peptide shown to protect against H.pylori and other pathogenic bacteria. Synthetic expression of active LL-37 in vivo is challenging due to the cytotoxic effects it has in the host. To make a resistant host that can export LL-37 to the media we intend to overexpress the E.coli acrAB and tolC operons, which activate the AcrAB-TolC efflux pump, a mechanism related with resistance to this and similar peptides by expulsion. To create a system in which E.coli expels LL-37 only in the presence of specific pathogenic bacteria, we use the LsrA promoter, which allows transcription in the presence of AI-2, a molecule produced by these bacteria to communicate via quorum-sensing. To avoid self-induction we designed an antisense RNA with specific secondary structure to inhibit the translation of LuxS, the enzyme responsible of the production of AI-2 in E.coli.

Team Colombia Uniandes: Nicko & Chimi: The magneto and the chimera

This year we are developing two projects: The first one, that we call Nicko, is an alternative solution for water pollution caused mainly by mining, it is a system capable to detect and absorb nickel, to later be removed magnetically, using parts of the homeostatic system of E. coli and Ralstonia metallidurans and the magnetotactic property from Magnetospirillum magneticum AMB-1 which will be used as our final chassis.

The second one, Chimi, is a stress-tester for animals (or even humans). It is based in a glucocorticoid sensor that is able to discern between basal levels and stress levels of glucocorticoid hormones in a sample with an easily recognizable signal, such as color, to allow the sensor to be used in the field, household or the laboratory.

Team Costa Rica Cibus: Genetic transformation of Bacillus subtilis for lactose consumption

Cibus 3.0 takes biodiesel production to a new level using dairy industry wastes. Annually, about 675 thousand tons of whey are thrown into rivers. This because at the present time there isn’t a program for reusing this waste, and producers find it difficult to treat them properly because of its chemical composition. Our idea consists in the modification of the bacteria Rhodococcus opacus to enhance the absorption of lactose and the overexpression of the natural triglycerides (TGA) producing ability of R. opacus, achieved by inserting an optimized sequence of a DGA acyltransferase gene and lactose absorption genes, constitutively expressed, also with an optimized sequence of a lipase from B. cepacia which is the responsible to break down the TGAs and an inducible “suicide device” in order to extract them with ease. Now all what it takes to finish the job is adding some ethanol to obtain our biodiesel!

Team Manaus Amazonas-Brazil: Electrobacter: from used frying oil to electricity

Used-frying-oil is produced in deep-fried food preparations and is one of the most serious environment hazards.In our project we are using the Shewanella which is a genus of proteobacteria widely found in Amazon region(called also as Shewie).They can reduce long-chain fatty acids, being a versatile new chassis to study and work in the iGEM competition. The fat acid degradation via Beta-oxidation is done by enzymes which expression is regulated by the genes FadR, FadL, FadD, FadE producing acetyl –CoA. All these features are remarkable for bioremediation of fat and oil spills.Besides that,is also known for its ability in “delivering” electrons to external media.we modified Shewie β-oxidation pathway silencing regulators and enhancing expression of some genes for fat degradation.In this years' project we aim to make a micro power plant using a bacteria hungry for used-frying-oil.

Team TecMonterrey: Modular, synthetic biology approach for the development of a bacterial cancer therapy in Escherichia coli.

By harnessing the inherent ability of facultative anaerobic bacteria to colonize and grow in tumoral environments, this project aims to prove the functionality of four different modules that would work together as a bacterial cancer therapy using Escherichia coli as chasis: Toxicity module, Secretion module, Localized induction module, and Internalization module. The expression of tumor specific therapeutic proteins, Apoptin and TRAIL, conforms the toxicity module. For these proteins to have their effect they need to be located in the extracellular matrix, therefore we are developing a module with a secretion function using hemolysin secretory mechanism. The hypoxic microenvironment present in tumors can be used for the localized induction module of tumor specific proteins, using the promoters HIP and nirB. Finally, Apoptin needs mechanisms to enter tumor cells’ cytoplasm. Proteins with this requirement could reach the cytoplasm when coupled with the internalization module, resulting in a fusion with the TAT peptide.

Team UANL Mty-Mexico: Integrating transcriptional and post-transcriptional regulation through the use of two synthetic RNA thermometers

Temperature sensing RNA sequences, known as RNA thermometers, regulate translation by preventing the ribosome from binding the transcript until higher temperatures shift it to an open structure. Several naturally occurring RNA thermometers have been described, and synthetic sequences that emulate them have been designed and proved to regulate genetic expression at different temperature ranges. Here, we intend to build a genetic circuit that results in three discrete states whose transition can be regulated by temperature changes only. Most notably, our circuit integrates transcriptional and post-transcriptional regulation, widening the spectrum of potential genetic circuit topologies for synthetic biology, with applications that range from basic research to the replacement of chemical inducers for industrial-scale processes.

Team UC Chile: Whateversisome: create your own bacterial functional organelle

Compartmentalization is a characteristic of complex biological systems. Carboxysomes are proteinaceous bacterial microcompartments that evolved to optimize bacterial metabolic reactions. We sought to take advantage of this biological principle to design a platform for in vitro metabolic engineering. Whateversisome it’s based on two hypotheses: the targeting signal to the microcompartment is present in a subunit of RuBisCO and that after isolation Carboxysomes can maintain their metabolic capacity in vitro. To address these hypotheses, we designed a system to target proteins of interest to the Carboxysome using RuBisCO subunits as targeting signals. We showed colocalization of GFP fusion proteins and Carboxysome shell-proteins fused to RFP. Second, we designed a simple system for Carboxysome purification based on biotinylation that should enable easy isolation of recombinant Whateversisomes. Our approach would enable combinatorial in vitro metabolic engineering by producing and combining arbitrary Whateversisomes. This project takes advantage of subcellular organizational principles for metabolic engineering.

Team UFMG Brazil: CardBio (Cardiovascular disease biomarkers sensor)

Death by heart diseases is very common worldwide, being Acute Coronary Syndrome (ACS) its main cause. This fact is deeply related to late diagnosis, which is usually made after the cardiac event had already occurred. We, from UFMG team, decided to explore this problem building a system capable of providing a precocious diagnosis for ACS based in 3 biomarkers: Brain Natriuretic Peptide (BNP), Trimethylamine-N-Oxide (TMAO) and Ischemia Modified Albumin (IMA). The main goal is to detect each of these biomarkers using our engineered E. coli by integrating the signals CFP, YFP and RFP produced when BNP, IMA and TMAO, respectively, are present in a sample of patient serum. This diagnosis is based on color intensity of the fluorescent proteins. So, we can establish the presence or absence and severity of ACS disease and predict earlier a myocardial event, thus providing information for fast treatment.

Team USP-Brazil: Detecthol: Methanol detection system

Our product is a bioengineered sensor, which will be able to detect levels of methanol above 2% in common alcoholic drinks. This will allow government to make high-throughput screening of ethanol drinks tainted will methanol. The device will be used as an initial low cost and portable test. The construction is based on the pAOX promoter, which is activated by methanol and repressed by ethanol. Several parts of the device must be tuned for proper function in Pichia pastoris; pAOX promoter, red fluorescent protein (RFP), Mxr1p transcriptional factor and FLD promoter. Since we aim to develop a product ready for the consumer to use, we plan to develop a plastic container for the lyophilized yeast, printed by 3D printer, that will help perform the test and will also contain the yeast. After use, the container will be able to apply bleach to eliminate the yeast.

NORTH AMERICA

Team Alberta: The Littlest Mapmaker

Inspired by a 2007 iGEM joint project by Davidson College and Missouri Western State University, 'The Littlest Mapmaker' is the University of Alberta's effort to create a biological computer capable of solving the Travelling Salesman Problem, a logistical challenge in which a hypothetical salesman must find the shortest route through a series of destinations. Our travelling salesman computer is built from a combination of raw DNA chemistry and bacterial colonies: first it assembles the routes by stringing genes together, treating them like roads on the salesman's map, then the bacteria sort the good routes from the bad, identifying the answer through the quantity of bacterial colonies making use of each route. The most commonly used route is the winner!

Team Arizona State: BactoVax: A Modular, Bacterial-based Cancer Vaccine Platform

Cancer kills eight million people each year, a number unchanged over the past five decades. The current paradigm for cancer treatment involves non-specific therapies such as chemotherapy and radiation that cannot differentiate between cancerous and healthy tissue. We propose a novel vaccine delivery system of tumor associated antigens and immunomodular agents encapsulated within probiotic bacteria to harness the patient's own immune system to fight cancer. The bacterial vaccine should activate macrophages and dendritic cells in order to teach the immune system to recognize cell surface antigens that distinguish tumors from healthy tissue. We aim to engineer lab-strain Escherichia coli and E. coli Nissle 1917, a commercial probiotic, to train the immune system to target and destroy tumor cells. This provides a distinct advantage over current bacterial vaccinology platforms, which rely on pathogenic bacterial chassis such as Salmonella and Listeria.

Team Berkeley: Genes to Jeans: a green solution to blue denim

The world consumes over 40 million kilograms of indigo annually, primarily for dyeing denim. Indigo is currently derived from petroleum using a high energy process, and commercial dyeing involves the use of reducing agents to solubilize the dye. The development of biosynthetic and bioprocessing methodologies for indigo dyeing could have environmental and economic advantages. By combining the biosynthesis of indigo and the use of the natural indigo precursor indican, we propose a more sustainable dyeing method as an alternative to chemically-reduced indigo in the large scale production of indigo textiles. We achieved in vivo indigo production in high titers, and efficient cleavage of indican using a non-native glucosidase. Inspired by natural systems, we isolated and characterized several plant and bacterial glucosyl transferases hypothesized to produce indican. Lastly, we compare the cost and environmental impact of our alternative with the present chemical process.

Team BostonU: Fuse, or Die: The Case for the MoClo Revolution

In order for synthetic biologists to be able to use automation technologies, we need a well-characterized library of basic biological components that can then be used to design more complex systems. MoClo is a one-pot digestion-ligation assembly technique developed by Weber in 2011, which enables faster and more efficient construction of genetic circuits when compared to BioBricks, the current iGEM standard. With our project, we are proposing that iGEM teams replace the inefficient BioBricks format with MoClo. We have expanded our library of basic MoClo DNA Parts and characterized devices using various promoter-RBS combinations via flow cytometry. We also designed and implemented a standardized data sheet with a JavaScript software program in order to easily share our library and data with the community. Our MoClo library, characterization data, and data sheet tool fill an essential role in the implementation of automated synthetic biology protocols.

Team British Columbia: CRISPR MADE BY U – CRISPR Mediated Automated Design Employed to Bring You Ultrabiotics

The past decade has seen the emergence of robust bioprocessing strains engineered to synthesize discrete molecular products. The next-generation of strains could be “programmable,” with on demand generation of molecules within a bioreactor e.g. a yogurt fermentation capable of making any combination of flavouring, nutrients or pharmaceuticals. While merging all this potential into single hosts seems efficient, it would also bring added risk in the case of a process failure due to bacteriophage infection. Here, we not only rationally design widespread immunity to phage infection, but also hack this immunity system to yield programmable biosynthesis at the community level. We demonstrate this by building both broadly and specifically neutralizing CRISPR systems that were paired with biosynthetic capabilities for vanillin, caffeine and cinnamaldehyde production. Eventually, a fermentative process could exist that is vaccinated to phage infection but susceptible to targeted phage addition that results in a programmable probiotic – or ultrabiotic.

Team BYU Provo: Phage Pharming: Two Approaches to Expanding the Use of Bacteriophage in Synthetic Biology

Bacteriophages are the most abundant organism on the planet, yet most are still uncharacterized. Current research is focused on finding new ways to use bacteriophage either in their wild-type state or after they have been modified for use in synthetic biology. We studied two ways to modify existing well-characterized bacteriophages. First, we employed random mutagenesis, CsCl purification, and plaque-size selection to isolate T4 and T7 bacteriophage with altered capsid sizes. A library of capsid sizes will allow researchers to select the appropriate bacteriophage for use in biotechnology or nanotechnology applications. Second, we designed a cholera sensing and destruction circuit using bacteriophage lambda. In this circuit, lambda contains biofilm-degrading enzymes controlled by a cholera quorum-sensing system transferred to E. coli. Upon sensing cholera, this E. coli will activate lambda, leading to cell lysis and biofilm degradation. This research demonstrates the versatility and utility of bacteriophages in the field of synthetic biology.

Team Calgary: The FerriTALE

Outbreaks of foodborne illnesses are a growing problem for food safety and public health. Whether in your water, salad or steak, pathogenic E. coli causes upwards of 250,000 illnesses every year. To solve this problem, iGEM Calgary is developing the FerriTALE to detect harmful E. coli. It uses engineered proteins that detect and report the presence of dangerous E. coli in a sample. The detector, TALE, binds to genomic markers specific to dangerous E. coli. Next, our TALEs are attached to the scaffold and reporter, Ferritin, that rapidly alerts the user to the presence of E. coli through a visible color change. We have integrated these proteins into a handheld device, similar to a home pregnancy test, that tells the user if dangerous E. coli is present. Moving forward, the FerriTALE can be tailored to detect other pathogens as the basis of a powerful new detection platform.

Team Carnegie Mellon: Light-Activated Antimicrobial Phage

Due to the widespread misuse and overuse of antibiotics, drug resistant bacteria now pose significant risks to health, agriculture and the environment. An alternative to conventional antibiotics is phage therapy. However, many temperate phage also form prophage. Our approach to antibiotic resistance is to engineer a temperate phage, Lambda, with light-activated production of superoxide. The fluorescent protein KillerRed was cloned into a plasmid vector and lambda gt11 with the IPTG inducible lac promoter. Lysogens were isolated and these strains were characterized and compared to E. coli with KillerRed from high-copy plasmids. Light activation of KillerRed resulted in decreased cell numbers. In addition, we modeled our system at multiple scales, including populations of phage and bacteria, KillerRed gene expression, ROS production, and effects of light. Having two methods of killing, lysis and superoxide, decreases the probability of developing resistance and our system overcomes the prior limitations of using wild-type temperate phages.

Team Clemson: Development of a Universal Self-Amplified (USA) Biosensor for Repaid Detection of Viable Pathogens

Many regulatory agencies such as the Department of Agriculture and the Environmental Protection Agency have specific standards for pathogen concentrations in sample materials, including “zero-tolerance” for some foodborne pathogens. However, current detection methods for these disease-causing bacteria suffer from one or more of the following limitations: 1) requiring sample enrichment, 2) inability of low-level detection, 3) indiscrimination between viable and non-viable cells, 4) small sample volume capacity, 5) tedious procedures, and 6) high assay cost. Our Universal Self-Amplified (USA) Biosensor uses a genetically modified detection bacteria to solve many of the aforementioned issues. The engineered USA bacteria will recognize a target chemical produced by the pathogen of study, which will trigger a cascade of genes to both amplify the chemical signal and produce a visible alert to the pathogen’s presence. The USA pathogen detection mechanism strives for rapidity, economy, and simplicity.

Team Concordia: Comput-E.coli

Our aim is to achieve universally computational cells through the exploitation of cell-cell communication to generate biological cellular automata. To achieve this, we are using an array of E. coli colonies, which implement the same logical functionality while using different input/output interfaces. The strains process their inputs (two inputs from neighbors plus their own current state) to decide what their next state will be, after the application of a global clock.
At the heart of our project is the construction of the clock, to synchronize information processing. We are making a fusion protein of ETR1 from A. thaliana and EnvZ from E. coli to allow cells to respond to ethylene gas concentrations. All cells will produce this gas at cyclic intervals, thus creating a truly autonomous clock. We have also devised an RNA-based system that can process information reliably and expediently with the use of ribozyme-based XOR and AND gates.

Team Cornell: Organofoam: Genetically Engineering Fungal Mycelium for Biomaterials Development

The goal of Organofoam is to develop a fundamental toolkit of genetic parts for engineering complex fungi, particularly plant-pathogenic basidiomycetes. We were inspired to do so by a local company, Ecovative Design, that uses lignin-degrading fungi and plant matter to produce a biodegradable Styrofoam substitute. The existing product that we are seeking to improve, known as “mushroom packaging,” is a sustainable and necessary alternative to Styrofoam. Polystyrene can take hundreds of years to degrade in landfills, produces dozens of identified chemical toxins upon combustion, and is tremendously inefficient to recycle, thus posing difficulties for disposal and polluting the environment. However, the production efficiency of Ecovative’s substitute suffers due to contamination from pathogenic molds, a problem that we seek to solve using synthetic biology. Using the complex, plant-pathogenic basidiomycete, Ganoderma lucidum, as a chassis, we are expanding the accessibility of fungal genetic engineering and demonstrating its utility for commercial purposes.

Team CU-Boulder: Cheap protein and DNA purification methods for DIY Bio

The focus iGEM at CU-Boulder has been to make synthetic biology more accessible and affordable. We spent the summer developing parts, procedures, and documentation to help make this vision a reality. The original goal was to create the constructs and purification methods necessary to produce and isolate restriction enzymes. Along the way we explored some novel approaches to DNA and protein purification and developed experimentally tested protocols for these and other procedures essential to Biobrick assembly. Our purification methods exemplify the ideal of using common lab materials to make performing everyday lab techniques as accessible and inexpensive as possible. A related aspect of our project was exploring methods of recycling consumables associated with lab work in order to reduce waste and material expenses. We hope that our findings using this 'do-it-yourself' approach of synthetic biology help make this type of research more accessible for those where funding is limited.

Team Duke: Designing Synthetic Gene Networks Using Artificial Transcription Factors in Yeast

Synthetic gene circuits have the potential to revolutionize gene therapies and bio-industrial methods by allowing predictable, customized control of gene expression. Bistable switches and oscillators, key building blocks of more complex gene networks, have been constructed using naturally occurring and well-characterized regulatory elements. In order to expand the versatility and variety of these circuits, we designed and constructed gene networks using artificial transcription factors (ATFs). The ATFs are of two classes: inhibitory TAL proteins and a catalytically inactive dCas9 protein with small guide RNA elements, each orthogonal to the yeast genome. Using mathematical modeling, we determined the parameters expected to create bistability and oscillation, using tandem binding site kinetics to achieve cooperativity. Based on these results, we assembled a library of plasmids containing ATFs, binding sites, regulatory elements, and fluorescent reporters. We then integrated these genes into the genome of Saccharomyces cerevisiae and are currently characterizing them using flow cytometry.

Team Gaston Day School: Fluorescent Detection of Cadmium in Water Supplies

Heavy metal contaminants pose a serious health threat around the world, especially in locations with poor irrigation. Cadmium, in particular, is a known carcinogen that affects the cardiovascular, gastrointestinal, and respiratory systems. The Agency for Toxic Substances and Disease Registry compiled a Priority List of toxic substances, on which Cadmium was seventh. We combined the Green Fluorescent Protein coding region with a Cadmium Sensitive Promoter to create our detector, which provides a simple and inexpensive test for the presence of Cadmium in water supplies. We will incorporate sensitivity tuners to decrease the detection threshold, and we will use mutagenic PCR on both the promoter and the entire detector to increase its sensitivity. Proper use of this BioBrick could result in early detection of cadmium polluted water and potentially prevent deaths worldwide.

Team Georgia State: Mamba Juice: Expression of Exogenous Mambalgin Peptide Using the pGAPzα Vector System

Pharmaceutical companies have invested considerable financial resources in developing analgesics. Often, the compounds used in these medicines are naturally occurring, such as aspirin and opoids. In 2012, Diochot et al from the Institut de Pharmacologie Moléculaire et Cellulaire in France, was able to extract and isolate an analgesic peptide found in the African Black Mamba. The peptide, Mambalgin-1, has been shown to have comparable analgesic effects to morphine but does not induce the debilitating withdrawal affects. However, extracting this peptide is expensive and dangerous work. Using a cassette of standardized systems (the pGAPzα expression vectors) in P. pastoris, we sought to produce high quantities of pure, functional proteins in a safe and cost effective manner.

Team GeorgiaTech: Bacterial BioBots: Integrin-Based BioSensors

Our team goal is to develop novel bacterial BioBots that respond to the extracellular tissue environment. Mammalian cells communicate with the extracellular matrix (ECM) using heterodimeric cell surface receptors, called integrins, which can signal in a bidirectional manner between the cell interior and ECM. We aimed to express the integrin αIIbβ3 in E. coli cells. To promote dimerization of the integrin subunits, we attempted to optimize bimolecular fluorescence complementation of split GFP using surface display technologies. We cloned split GFP parts, assembling the T7-promoter, LacI-operator, and ribosomal binding site (RBS) upstream of the protein-coding region. To verify αIIbβ3 function, we developed an integrin activity sensor consisting of the ligand derived from fibrinogen (KQAGDV) coupled to GFP. Finally, we successfully created a new standard for RBS addition that inputs the strong RBS (BBa_B0034) in front of any standard BioBrick part, which is efficient and more successful than the usual 3A/standard assembly.

Team Greensboro-Austin: Bioadhesive Production Using an Expanded Genetic Code

Mussel adhesive proteins (MAPs) are water-resistant bioadhesives that have a variety of biomedical and construction applications. Replicating the natural properties of MAPs through in vivo production using microbes is difficult as it depends on post-translational modifications, primarily the hydroxylation of tyrosine residues to L-DOPA. Since adhesiveness is correlated with L-DOPA content, our project aims to improve the adhesive properties of MAPs by utilizing a strain which inserts the non-canonical amino acid L-DOPA at UAG stop codons during translation. When combined with the in vivo production of free L-DOPA, this system is a significant step towards rapid, cost-effective MAPs production. Our team also worked on degrading odorous compounds, computationally modeled the detection of potentially dangerous oligo orders, and created BactoArt using inducible fluorescent proteins. Additionally, we created the Open Sequence Initiative, which is focused on updating the standards for submitting BioBricks to the iGEM Registry.

Team Lethbridge: Frame-Changer: Shifting Translation for Multiple Protein Expression

The current growth in synthetic biology research promises more complex and useful engineered systems. However, increased complexity often requires more genetic material that can be difficult to introduce into organisms. We propose the development of a new library of regulatory gene expression elements that allow for compression of multiple coding sequences into a smaller amount of genetic space. Using a pseudoknot RNA structural motif, commonly used by viruses to minimize their genome size, we will show the utility of dual-coding gene sequences to give useful protein products whose expression can be regulated by the pseudoknot’s ability to induce ribosomal frameshifting. A software tool will also be used to overlap multiple coding sequences into different reading frames. Ultimately, this library of standardized parts will be available for use in a variety of engineered systems requiring minimal coding space and multiple protein expression.

Team Michigan: A Completely Unidirectional Biological Transistor Utilizing an Engineered Fim Switch

Recent studies have just started to explore the possibility of utilizing existing recombination systems, to store information and perform computations. However, the only systems studied so far are not completely unidirectonal in their ability to flip a segment of DNA. Instead, previous systems have relied on “recombination directionality factors”, which when complexed with the unidirectional recombinase, reverse the direction in which it flips the DNA segment. The fim system from E. coli, has been shown to contain 2 unidirectional recombinases, hbif and fime, which flip a promoter containing segment of DNA. Our project seeks to engineer the fim switch by replacing the native promoter with another promoter. We demonstrate that it can function as a reliable and efficient biological transistor, or “transcriptor”. Beyond storing information and performing basic computations, the system would serve as a very useful, tightly controlled switch.

Team Minnesota: The pMNBB vector system: A toolkit approach for engineering Pichia pastoris

Currently, there is a significant gap in available resources for transformation and expression of foreign proteins in the industrially-relevant yeast model, Pichia pastoris. To address this, we have constructed pMNBB, a versatile shuttle vector system. pMNBB vectors provide researchers a modular BioBrick platform to introduce synthetic pathways into E. coli and also the option to rapidly transfer these constructs into P. pastoris. Not only is this vector system designed for integration of expression cassettes into the P. pastoris genome, but we have also added elements which allow for the episomal maintenance of these plasmids. Moreover, these vectors are amenable to trans-kingdom conjugation (TKC). Rather than following a yeast transformation protocol that can take days to weeks, our constructs may be introduced to a yeast culture in roughly one hour. Finally, as a proof-of-concept, we use the pMNBB vector system to produce and secrete active human insulin from P. pastoris.

Team MIT: Engineered mammalian cell-cell communication mediated by synthetic exosomal cargoes.

Coordinating behavior across cell populations to form synthetic tissues requires spacial communication between individual cells. While there has been some success engineering single signals, sending multiple signaling elements spanning spatial scales for multicellular coordination remains a significant hurdle. Here, we describe a method for mammalian cell-cell communication utilizing engineered exosomes containing miRNA or protein signals. First, we demonstrate selectively packaging signaling miRNAs (miR-451 and miR-503) and synthetic fusion proteins (GFP, Cas9, and Cre recombinase each individually fused to the oligomerizing membrane targeting domain Acyl-TyA) into exosomes within cells engineered with sender genetic circuits. Next, we demonstrate that these miRNA and protein signals can modulate gene expression within cells engineered with receiver genetic circuits. Finally, we present preliminary cell-cell signaling results on populations of cocultured sender and receiver cells. Our method may enable multiplexed communication among populations of various cell types and the creation of sophisticated synthetic tissues.

Team MSOE Milwaukee: Synthesizing Eucalyptol from Spent Grain Waste using a Three E. coli System

Eucalyptol, the main component of Eucalyptus oil, is a valuable product with multiple potential industrial uses. Current methods are costly, which is a major deterrent for research on these applications. We have designed a system consisting of three strains of Escherichia coli (E. coli) which will convert spent grain waste from breweries into Eucalyptol. The design utilizes two strains of E. coli overexpressing and secreting enzymes to degrade hemicellulose into xylose. The third strain of E. coli will utilize the xylose and convert the molecule into Eucalyptol using the Mevalonate pathway. A scale-up model was developed to assess industrial feasibility. The cost of production would drop two-thirds, which would make industrial uses plausible. Future studies will be transforming our E. coli with the genes of interest and testing for production of Eucalyptol.

Team Nevada: Lysesavers: A novel endolysin-based bactericide for the treatment of gram-negative pathogens

Endolysins are the bacteriophage proteins responsible for lysing target bacteria by degrading the peptidoglycan layer. The use of endolysins to fight bacterial plant disease is an emerging field, but it has largely been limited to gram-positive bacteria, as the outer membrane of gram-negative bacteria prevents endolysins from accessing the peptidoglycan. Recombinant E. coli have been created to express endolysins which target various plant pathogens. These proteins can be used as a novel gram-negative bactericide when coupled with a treatment to disrupt the outer membrane. A system to easily detect outer membrane disruption will be critical to our project, as well as any future studies on gram-negative endolysins. To address this, a registered iGEM part used for cell surface detection was improved and incorporated into a fluorescence-based assay. Erwinia amylovora, a gram-negative bacterium responsible for fire blight in fruit trees, was chosen as a model target for proof of concept.

Team Northwestern: NU Balance

The purpose of this iGEM project is to combat oral diseases by engineering a bacteria to neutralize the lactic acid produced by Streptococcus mutans. Due to the time constraint, the scope of the project is limited to a dual-state promoter where an acid-responsive promoter is placed upstream of a constitutive promoter. This novel transcription regulation element will have both constitutive and inducible activity. The construct will be tested by linking green fluorescent protein to the downstream promoter. Based on preliminary results, the extra promoter should allow for a significant increase in gene expression. These pieces have wide implications in any fields that require a quick upregulation of any gene in response to acidic environments.

Team OU-Norman OK: A Shuttle Vector for Clostridial Chassis Organisms

Concerns about energy security, sustainability, and the environmental impact of fossil fuels have renewed the interest of both the public and the scientific community in the development of renewable energy sources, including biofuels. Recent research in synthetic biology has resulted in the production of alcohols not known to be produced naturally, and the extension of carbon chain length. These systems have been expressed in E. coli. Historically, the alcohol titer obtained with native alcohol producers, such as Clostridium acetobutylicum, an acetone/butanol/ethanol producer, and Clostridium beijerinckii, an isopropanol/butanol/ethanol producer, has been greater than that obtained wtih non-native producers. One major problem with these systems is the lack of tools available for genetically manipulating them. The goal of our project has been to develop a shuttle vector that will allow the heterologous expression of these and similar biosynthetic pathways in these organisms.

Team Penn: Engineering the Epigenome

The code of life is more than a sequence of A’s, C’s, T’s and G’s; epigenetic modifications, such as DNA methylation, are powerful and heritable regulators of gene expression. Targeted methyltransferases are enzymes that catalyze sequence-specific methylation – the most useful tool for engineering the epigenome. With a synthetic biology approach, we developed an assay to test targeted methyltransferases without expensive, time-consuming traditional methods. Our modular single-plasmid system allows methyltransferases to be easily cloned and tested via inexpensive digestion assays, quickly measuring the existence and extent of targeted methylation. Additionally, our plasmid contains standardized primer-binding sites for methylation-sensitive sequencing, and our E. coli chassis effectively eliminated noise associated with methylation studies. We are using this assay to characterize our novel targeted methyltransferases, which could be used to study epigenetic modifications. In the future, synthetic biologists could embrace these tools to explore the next frontier in engineering biological systems: the epigenome.

Team Penn State: Plants as Plants: Natural Factories Producing Fuel, Plastic, Flavoring, and More

Plants as Plants: Natural Factories provides a green approach to the manufacturing of valuable chemicals and materials. Through synthetic biology, we are able to control the expression of genes that regulate the production of desired secondary metabolites. Via the manipulation of established metabolic pathways, we hope to produce vanillin and butanol. The prospect of being able to synthetically produce a biofuel provides vast possibilities for the scope of synthetic biology and green energy. Additionally through the manipulation of the cellulose synthase genes, we hope to increase the biomass of plants by a hybrid plant cell wall. As shown through these projects, the use of plants provides various green energy possibilities. However, due to the limited use of plants within synthetic biology there are various regulation issues. Thus we have additionally worked on characterizing a range of plant promoters as well as introducing the Cas9 crisper system into plants.

Team Purdue: Back to the Basics

Synthetic biology has striven to prove that classical engineering principles are applicable in the field of biology. Several challenges have yet to be overcome including design of robust genetic circuits, reliable gene expression, and a standard way to characterize parts. To assess circuit robustness, we utilized the power of the Taguchi Method, a statistical analysis which optimizes a set of parameters to form a robust system against outside noise while minimizing experimental time and cost. Making bicistronic expression operating units, which reduce the variability of gene expression, available in the Registry of Standard Biology Parts will enable efficient engineering of large networks. Finally, collaboration among teams allowed for a new standardized form of submitting characterization of parts to the Parts Registry. These three approaches to improve part standardization and robustness will help move the field of synthetic biology one step closer to proving that biology can, in fact, be engineered.

Team Queens Canada: Biosynthesis and breakdown of human odour compounds for the behavioural manipulation of malarial mosquitos

Malarial mosquitoes are developing resistance to key insecticides and drugs, and are becoming diurnal to avoid treated mosquito nets. Recent studies have shown that the African mosquito uses human foot odour to locate its host, a trait that is enhanced when the insect is carrying malaria. We plan to combine a carboxylic acid reductase and an acetyl transferase, in order to create E. coli capable of converting a major component of foot odour (isovaleric acid) into banana smell (isoamyl ester). This could have both commercial and humanitarian applications. Our second goal is to deliberately synthesize mosquito attractants inside traps. Recent research has shown that a mixture of CO2 and foot odour volatiles can be more attractive than a human. We have chosen indole as our first attractant, a compound naturally found in human sweat. We hope our project will show that bioremediation and biosynthesis techniques have applications in mosquito control.

Team RHIT: Constructing a Unique Platform for Interspecies-Dependence (CUPID): The evolution of multicellular machines.

CUPID aims for stable obligate symbiosis for the model prokaryote Escherichia coli (bacteria) and the model eukaryote Saccharomyces cerevisiae (yeast). We address this goal by constructing a unique platform for interspecies-dependence based on inducible expression of a required histidine biosynthetic gene (HIS) in each species. Expression of the bacterial HIS gene is induced by lactic acid produced by constitutive expression of a lactate dehydrogenase gene in the eukaryote. Expression of the yeast HIS gene is induced by the binding of its mating factor receptor to mating factor expressed constitutively on the surface of the prokaryote. Histidine deprivation necessitates physical contact and symbiosis for survival. Such pressure may facilitate evolution of a stable exosymbiotic form of the two species. Study of isolates and further manipulation could provide insight into the use of obligate symbiosis in synthetic biology and yield a chassis for the synthesis of novel multicellular machines.

Team Rutgers: Sensteria: Quorum Sensing E. Coli

Our project aims at developing a self-regulatory system for the degradation of virulent factors. We plan to integrate quorum sensing and pon1 to create this system of self-regulation. Quorum sensing will be used to detect the presence of high cell densities and to activate pon1 upon detecting high cell density. Pon1, acting as a repressor, will degrade the signal molecules needed for quorum sensing and thus turn off quorum sensing as well as itself. It will reactivate upon the reactivation of quorum sensing and this cycle of self-regulation will persist. This prototype system is meant to test the application of this system but the main goal is to introduce this system to pseudomonas aeruginosa as a means to prevent the harm that it causes.

Team Stanford-Brown: Synthetic Bio-Communication

Communication is a dynamic requirement for life as we know it. We are using cellular and molecular messaging of different magnitudes to improve the broadcasting and reception of information. Starting on the atomic level, our BioWires project has created silver-incorporating DNA to act as nanowires, which could improve the cost and effectiveness of electronic products. Our CRISPR project is creating a system for DNA messages and resistances to be passed from cell to cell, in effect, creating transmissible probiotics and changing the way that cells communicate. We are also building a chromogenic biosensor to detect sucrose secretion that will be launched on a satellite (EuCROPIS) into low-Earth orbit. Finally, our De-Extinction project involves decoding messages from the past to better understand early life on Earth.

We are the Stanford-Brown iGEM team, and we're connecting life on Earth, to help us prepare for life beyond it.

Team Toronto: Biofilm: System engineering in E. coli

Microorganisms frequently adopt a lifestyle in which they excrete extracellular biopolymers to aggregate and form biofilms. We are researching the pathways that induce biofilm formation and maturation in E. coli, for modulating surface-specific adhesion of E. coli biofilms. We are constructing and characterizing E. coli strains with targeted deletions or recombinant protein expressions that are crucial in biofilm formation pathways. In response to environmental stimuli such as temperature, blue light, or sodium, the phenotype of each mutant E. coli strain will be characterized using a specially developed high-throughput protocol. Biofilm formation control has applications for bioremediation, in which we are pursuing in a related project on heavy metal precipitation. Another potential application is in medical treatment of pathogenic infections, since the generally slow diffusion rate in biofilms gives pathogenic bacteria/fungi an additional mechanism for antibiotic resistance phenotypes.

Team UC Davis: RiboTALe: A Tunable and Modular System for Control of Gene Expression

Despite the fact that the Registry of Standard Biological Parts contains a large number of inducible promoters, the actual usage of these parts is dominated by a very select few. In order to increase the versatility of expression control systems, we propose a new system coupling transcription activator-like effectors (TALEs) with inducible riboswitches. TALEs are proteins secreted by the bacterial pathogen Xanthomonas that contain engineerable, sequence-specific DNA binding domains and can act as transcriptional repressors or activators. We plan to manipulate TALE activity by subjecting them to inducible expression through riboswitches and promoters. By pairing TALEs with riboswitches, we can expand the existing library of inducible repression systems. In addition, we hope to modify the parameters of our system to show the tunability and modularity of our overall construct. Through proper characterization, we believe that iGEM teams may also use these modular repression systems for the development of future devices.

Team UChicago: Keratinase Expression System in E. coli and B. subtilis

Each year, the poultry industry produces over two billion pounds of feather waste that is mostly processed into nutrient-poor animal feed but recent research has shown that feather keratin can be used to produce biodegradable plastics, fertilizers, detergents, and pharmaceuticals. However, current chemical methods for keratin degradation are energetically costly and previous efforts at keratinase production in heterologous hosts have been stymied by poor protease expression. To address both these problems, our team constructed BioBricks based on kerA, a serine keratinase gene native to B. licheniformis active on whole poultry feathers. We designed two biobricks for expression in E. coli and constructed a high copy number BioBrick plasmid with an origin of replication compatible with B. subtilis. By designing a new keratinase expression system in B. subtilis, we hope to provide a faster, cheaper alternative to current methods of industrial keratinase production.

Team UCLA: DiversiPhage: Library Generation for Protein Selection

Both the mammalian immune system’s complex defenses and a bacteriophage’s targeting mechanism depend upon protein diversification. These models have inspired innovations ranging from targeted drug delivery to protein display. Using the major tropism determining (MTD) protein expressed on the Bordatella bacteriophage BPP-1, we aim to develop an in vitro system for generating antibody-like proteins that bind specified targets. The MTD protein expressed at the phage’s tail fiber is naturally modified at its variable region to produce nearly 10e13 possible binding variants while preserving its structure. Mutating the MTD’s variable region by PCR can match the massive diversity of MTD in vitro. A library of MTD protein-DNA fusions generated by mRNA display can then be screened for binding against specified protein targets. This in vitro analog to phage display and immune clonal selection can be a powerful tool for constructing target-binding MTD variants with equally many varied applications.

Team UCSF: Operation CRISPR: Deploying precision guided tools to target unique species in a complex microbiome

In microbial communities, bacterial populations are commonly controlled using indiscriminate, broad range antibiotics. There are few ways to target specific strains effectively without disrupting the entire microbiome and local environment. The goal of our project is to take advantage of a natural horizontal gene transfer mechanism in bacteria to precisely affect gene expression in selected strains. We combine bacterial conjugation with CRISPRi, an RNAi-like repression system developed from bacteria, to regulate gene expression in targeted strains within a complex microbial community. One possible application is to selectively repress pathogenic genes in a microbiome, leaving the community makeup unaffected. In addition, we use CRISPRi to lay the groundwork for transferring large circuits that enable complex functionality and decision-making in cells.

Team UGA-Georgia: Geraniol production via novel protein expression tools in Methanococcus maripaludis

Geraniol is an intriguing 10 carbon compound with diverse applications including use as an agent for cancer prevention, fragrance, insect repellent, proposed biofuel etc. We explored and engineered a novel gene expression tool (BBa_K890000) for Methanococcus with the capability of expressing geraniol synthase from Ocimum basilicum (BBa_K1138000). We report the biosynthesis of geraniol at over 5% of DCW by transforming the vector into Methanococcus thereby expanding its native isoprenoid pathway. Furthermore we engineered new vectors (BBa_K1138001 & BBa_K1138002) with the potential capability of regulating and quantifying the expression of desired proteins via red fluorescence. This work demonstrates the use of Methanococcus as a cell factory for chemical production and highlights synthetic biology advancement by engineering new systems over traditional biological systems such as Escherichia coli.

Team UIUC Illinois: Cardiobiotics - A Genetically Engineered Approach to Cardiovascular Health

Cardiovascular disease (CVD) has been the leading cause of death in the United States for over twenty years and is a rising global health issue. Recent studies demonstrate a correlation between CVD and atherosclerosis, the buildup of plaque in the arteries. One associated risk for atherosclerosis is the production of Trimethylamine N-oxide (TMAO) by natural gut flora when metabolizing L-carnitine, a chemical found primarily in red meat and energy drinks. We created a probiotic to attack the root of this problem by outcompeting the gut bacteria for L-carnitine in order to suppress the production of TMAO. L-carnitine transporters (caiX and cbcWV) and L-carnitine dehydrogenase (CDH) derived from Pseudomonas aeruginosa were expressed in a safe strain of E. coli (Nissle 1917). This engineered E. coli can uptake and metabolize L-carnitine along an alternative, safe pathway into 3-dehydrocarnitine. Together, this system offers a novel solution in preventing TMAO-related cardiovascular health conditions.

Team uOttawa: Fold-change molecule detection using the Type-I Incoherent Feedforward Loop

Many synthetic gene networks are susceptible to cellular noise, as they rely upon the absolute levels of gene regulators which can vary greatly between individual cells. To address this, uOttawa has engineered a network in S. cerevisiae that is responsive to fold-changes as opposed to absolute changes in stimulus. This allows the network to maintain sensitivity despite noise, and also permits response to stimuli in a much larger dynamic range. By modifying the promoter driving the stimulus, the network can be engineered to detect fold-changes of any molecule with a responsive promoter, thereby serving as a structural chassis for the next generation of molecule detectors. In addition, we have also authored a children's book aimed at disseminating the concepts of synthetic biology to the public, and have designed an online interface that will facilitate the rapid construction of devices built from the Registry of Standard Biological Parts.

Team UT Dallas: A Multifaceted Approach Against Tooth Decay

Cavities are a problem faced by people and animals worldwide and are primarily caused by the bacteria Streptococcus mutans. S. mutans digests certain sugars, consequently forming lactic acid that can lead to tooth decay. Our system starts with the detection of saccharides essential for S. mutans’ survival. Using dextranase, the E. coli are able to attach onto the dextran layer that S. mutans normally live on. This allows the E. coli to deliver a targeted dose of norspermidine, helping with the breakdown of S. mutans biofilms. We also took another approach that utilizes a natural quorum signaling molecule, competence stimulating peptide (CSP). This peptide is responsible for population control in S. mutans. By using these approaches, we hope to aid in the treatment of cavities in both humans and animals.

Team Utah State: AMPed up E. coli

Antimicrobial peptides (AMPs) are peptides that have activity against a wide range of microorganisms. AMPs can vary greatly in their size, structure, and mode of antimicrobial activity. Due to the immense interest in both testing and characterizing AMPs (from various forms of life) a sustainable method to produce and purify them is necessary. Using Synthetic Biology as a platform, the production of AMPs in E.coli could potentially offer a cost-effective approach for large-scale peptide manufacture, as opposed to isolation from natural sources. After successfully manufacturing several AMPs using E.coli, these peptides will be tested on a wide variety of organisms to screen for antimicrobial activity.

Team UTK-Knoxville: Modular Design of Chimeric Biosensors

The major limitation in synthetic biology today is the lack of numerous, well characterized sensors. Our project aims to provide a reliable scaffold to test potential sensing domains with unknown substrates. We have created a standard platform to test a range of intracellular and transmembrane domains. Positive results are reported with red fluorescent protein for easy identification which can be done with high throughput methods such as 96 well plates and flow cytometry. We test our platform on sensors with interesting known responses. The chimera proteins are also useful in creating signals orthogonal to the cell.

Team Virginia: Minicells: Multi-Purpose Nano Chassis

Overexpression of the tubulin-homolog FtsZ leads to asymmetric cell division in E. coli that yields achromosomal 'minicells.' The lack of a chromosome renders minicells unable to replicate and cause infection, yet they still retain and express plasmid genes. Furthermore, minicells inherit the stable, non-leaky membranes and cytosolic composition from their parent cell. Our project design is centered on the creation of an IPTG-inducible FtsZ Biobrick that permits tunable overexpression for optimal minicell production. With the development of a multi-purpose, innocuous bacterial chassis as our ultimate goal, we incorporated three additional safety elements: the Ail protein, a polysialic acid capsule and de-acylated lipopolysaccharide. Both Ail and the PSA capsule serve to prevent complement deposition on the surface of the minicells, with PSA also protecting against antibody opsonization. Finally, LPS toxicity is reduced by inducing minicell formation in an lpxM mutant strain that lacks a critical myristoyl transferase for late-stage acyl modifications.

Team Washington: Red Light! Green Light!

Refinement of functional systems is a key aspect of engineering. Biological systems are not immune to this fact and must be continuously improved to function consistently, and reliably. Creating easy methods for tuning biological systems using light was the goal of this year's effort. Our team sought to improve on a previous light-regulated gene expression method, integrate the use of tablet device for testing, and create multiple functional biobrick constructs. In 2012, we developed an app that affords any researcher with access to an android device with a LED screen the ability to illuminate cells in a controlled manner for synthetic biology applications. We improved the functionality of the app by adding a mini petri dish setting and demonstrated the advantages of paralization by performing experiments in a 96 well plate format. This tablet application represents the first real life example of using a mobile device as synthetic biology instrument.

Team WashU StLouis: Converting E. Coli into a Nitrogen Bio-Fertilizer Using a Cyanobacterial nif Cluster: an iGEM project

The nif cluster of Cyanothece 51142 consists of 29 genes that construct and regulate a nitrogenase protein complex. This year, our iGEM team worked on harnessing the power of nif to produce ammonia in Escherichia coli. After synthesizing a nif-containing plasmid (28 kbp) using the DNA assembler method (Shao et al 2009) and transforming that plasmid into E. coli, our team tested for nitrogenase activity using the acetylene reduction assay. The transformed E. coli were then compared to wild-type under limited nitrogen conditions to check for a competitive advantage. Tests were used to evaluate the expression of various nitrogenase subunits. Our team also aimed to further characterize the promoter sequences of the Cyanothece 51142 nif cluster. Between the cysE and nifB genes, there is a 958bp uncharacterized, bidirectional promoter region of particular interest. We used fluorescent reporters to identify key regions within this promoter sequence under various environmental conditions.

Team Waterloo: Controlled Modification and Intercellular Transmission of a DNA Message

In nature, intercellular communication allows coordinated cellular behavior on a population level. Engineers seeking complex programmed population-level behavior require tools enabling controlled, information-rich intercellular messaging. Given its versatility and universality as an information-encoding molecule, DNA suggests itself as a message-carrying molecule to enable information-rich messaging. The Endy group at Stanford recently published a proof-of-principle demonstration of such DNA messaging wherein a DNA message was transmitted from one bacterial population to another carried by M13 bacteriophage particles. Here we propose an intercellular communication program that extends DNA messaging by controlling modification and transmission of a DNA message. Modification is controlled through flipping a DNA switch on the message DNA – a promoter sequence that is invertible using serine integrases and recombination directionality factors (RDFs). Transmission is controlled by placing expression of the M13 major coat protein, which is required for viral packaging of message DNA, under control of such a switch.

Team Wellesley Desyne: Enhancing Bio-Design with Next-Generation Human-Computer Interaction

Systems that integrate the wide array of technological tools available to synthetic biologists are needed more than ever. As the field of synthetic biology continues to advance, it is critical to communicate the applications, goals, and limitations of synbio research to the public. Our team is creating a software suite, which addresses technical synbio challenges while improving end-user experience and harnessing human-computer interaction (HCI) to engage the public in synbio concepts. Eugenie is a visual language and integrated development environment for Eugene that allows biologists to specify biological parts, properties, and device composition rules. zTree is a tool for visually representing the Registry of Standard Biological Parts to support sense-making of complex, hierarchical data sets. Bac to the Future is a web-based, interactive application that utilizes Twitter to illustrate synbio ideas to the public. The application of HCI techniques to synbio fosters more effective, collaborative, and intuitive software tools.

Team WLC-Milwaukee: The One Ring to Secrete Them All

Our construct utilizes removable purification and secretion tags separated by a single enzyme cut site NheI that is the insertion point for any coding sequence of interest. The induced gene can be secreted from the cytoplasm to the extracellular space via a tripartite secI pump. A cymR-regulated T5cumate promoter within the plasmid controls a Tse2 toxin to avoid horizontal gene transfer, while the plasmid also encodes a chimeric ompA-monoclonal antibody gene, which when expressed, inserts into the outer membrane and the antibody selectively binds norovirus within the gut to hinder infection. This plasmid construct was used to over-express and secrete plant-based, polymer-degrading enzymes bglS, yesZ, and xynA isolated from the cellulosome of Bacillus subtilis subtilis 168 to digest plant material. We utilized Escherichia coli Nissle 1917 containing a chromosome-integrated cymR gene to secrete these enzymes in the hope to accentuate dietary uptake for humans and livestock in impoverished countries.

Team Yale: Converting E. coli into a foundry for bioplastics

Poly(lactic acid) (PLA) is a biodegradable, biocompatible, bioresorbable, thermoplastic bioplastic that offers many advantages over other biomaterials in both commercial and medical applications. The current chemical method of synthesizing PLA is expensive, and the required processing and purifying steps use many environmentally unfriendly chemicals. Recently, E. coli has been engineered to produce PLA, but low yields and short chain lengths prevent the approach’s commercial use. Here, we evaluate the potential of using multiplex automated genome engineering to raise both yields and chain lengths of biosynthesized PLA by directing the E. coli metabolism to funnel resources toward PLA production without sacrificing the organism’s viability. Efficient biosynthesis of PLA, which may be thus achieved, would be a significant step in reducing the impact of plastic waste, and would benefit those receiving bone implants. It would also open up a new possibility in rapid manufacturing of personalized bone implants using three-dimensional printers.