Talk:Results

Wave pattern: Our first project was to create a pattern by the change of colors (by production of different fluorescent proteins) triggered by a signalling protein in a concentration dependent manner; so that the production of a protein as a result of the processing of a separete system, would trigger the pattern formation, just like a goal triggering a Mexican wave in soccer games. The idea was to have a plate having a lawn of cloned E. coli cells having 3 constructs. Each construct would have a promoter, which would be activated by a different level of the initial signal protein, and a coding part for a reporter fluorescent protein. In the figure below, red is represented to be the most sensitive reporter, activated by the lowest concentration. Yellow is sensitive to only a higher concentration and green requires the highest concentration. The triggering protein (say protein A) would be dropped at the middle of the plate, creating a concentration gradient on the plate as it diffuses. According to this, the color change woulld be for once, and as concentration of A rises in time first at the middle then towards the edges, the colors will alternate from red to yellow to green, and eventally all plate will become green.
Chase Simulator: The idea of project was to simulate the competition between two different warrior cell types made from E. coli to invade a semi-solid plate having a lawn of passive cells. The green and red cells are the invaders, and they represent two teams. The 'empty cells' are the ones to be invaded. Empty cells have two constructs and each code for a fluorescent protein and an inhibitor. They are normally off. One of the constructs (coding for green) can be activated by nearby green cells and the other(coding for red) by nearby red cells, possibly through quorum sensing molecules. Once one of the constructs in the empty cells is activated by an invader, expression of this construct irreversibly prevents activation of the other and the cell becomes permanatly green or permenantly red. The aim was to have a sort of game, where red cells and green cells would be competing to convert to empty cells so that they would not affected by the opponent cell any more. * The game could be fun, if 2 people separetely inoculate an area of their own choice on the same plate having a lawn of empty cells, with same amount of green and red invader cells each. The winner could be determined after incubation, by using fluorescence to see whose cells have invaded more of the plate. To add some challange, plates with different topologies (mountains or seas could be made ont the agar plate) could be prepared so choosing the area of inoculation would become a strategical decision. * The important point in this project is to have two promoters which are normally off, and also repressed and activated by distinct proteins. There should be no leakage, because a little leakage in one of the constructs inhibits the other irreversibly. As there are not many of such non-leaking promoters, we created a less complex version

Fast Ubiquitin assays: The search for renewable energy sources from alternative crops has led to the desire to understand the pathway by which lipids are synthesized. Higher lipid content has a direct correlation to higher energy value, so if the plant pathways involved in lipid metabolism can be modulated, biofuels could become more economically feasible. It has been reported that the ubiquitin proteasome pathway affects lipid metabolic processes. The ubiqination of target proteins for degradation requires the sequential activity of three enzymes: a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), and a ubiquitin ligase (E3). The objective of the 2007 MSU iGEM project is to develop a rapid and sensitive method for assaying the ubiquitin ligase (E3) activity. The E3 activity is generally determined by using an in vitro ubiquitination assay. The E3 is first incubated with ATP, ubiquitin, and wheat germ lysate containing E1 and E2 activities (or purified E1 and E2). The ligase reactions are then fractionated by electrophoresis on an SDS-PAGE gel and subjected to Western blotting for the detection of high molecular weight polyubiquitinated E3 proteins. This detection method is costly and time consuming. To develop a quicker assay of E3 activity, the E3 coding region is cloned into a plasmid and expressed as an in-frame C- (or N-) terminal fusion with a GFP protein. The E3 activity is then assayed by directly irradiating the protein gel with long wave ultraviolet light. The expression of green fluorescence in multiple, high molecular weight protein bands would indicate the ligase activity. To confirm that the multiple protein bands are ubiquinated, a plasmid containing RFP in frame with ubiquitin will also be constructed and transformed into E. coli cells harboring the E3-GFP fusion. Future work will involve in designing universal plasmids to provide for a quicker assay of any E3 ligases.

Cellular mercury purification Mercury contamination of drinking water is a significant problem in both developed and developing countries. Techniques to filter it out are both costly and intensive. Thus, the MIT iGEM 2007 team is engineering a biological mechanism to cost-effectively sense and remove Mercury ions from contaminated water through a two cell system. One cell will use the Mer promoter to sense the presence of Mercury ions, then activate the GFP fused downstream. The other uses a cell surface display mechanism to exhibit a Mercury capturing peptide, extracting the Mercury from the water. Both cells also display polystyrene binding peptides, and will thus be attached to a polystyrene filter. This setup would be easy to use, cheap to manufacture, and economical to distribute. It could be used from very small scales to even an entire village's drinking water supply.

GlucOperon: Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality.

Bottom up Biology: A proof of principle: Our project is designed so that, by measuring the characteristics of relatively simple devices called 'open-loops', we are able to predict the responses of more complex systems called 'closed-loops'. These predictions can be directly tested against the observed responses. If we are successful, it would represent the first experimental validation of this powerful bottom-up design principle. Open loop: This is a a black box with a parameter knob (blue), an input (yellow) and an output (cyan). Keeping the parameter fixed, we can dial up the input and measure the resulting outputs, giving us the open-loop characteristic (purple curve). Equivalence: This tells us precisely how much output corresponds to a given amount of input, shown as the equivalence line (gray line). Closed loop: This is a black box with a parameter knob (blue) and a state (yellow). We can vary the parameter values and measure the resulting state, giving us the closed-loop response. The point where the open-loop characteristic intersects the equivalence line is a self-consistent state: the given output, when fed back through the system, returns precisely the same output. At this point, the open-loop and closed-loop systems look identical, and the input of the open loop matches the state of the closed loop. By measuring these intersections as the parameter is varied, we can predict the closed-loop response from open-loop characteristics. We used three transcriptional regulatory modules to build our loops: the IPTG-inducible lac system, the aTc-inducible tet system, and the lux quorum-sensing system of Vibrio fischeri. The measured response of our constructs can be found in their datasheets. The key measurements are the following:
* Open-loop characteristic: [Sen-TIC+Rec-LRY.RC]
* Equivalences: [Trc-LC], [Trc-LRY]
* Closed-loop response: [Sen-TIC+Rec-RRY]
The open-loop characteristic [Sen-TIC+Rec-LRY.RC] shows the CFP level (output, cyan), as LuxI.CFP (parameter, blue) and LuxR.YFP (input, yellow) are varied. Equivalences, relating the amounts of CFP (cyan) and LuxR.YFP (yellow), can be determined by comparing [Trc-LC] and [Trc-LRY]. We used these measurements to predict the closed-loop response, and compared it against the measured response [Sen-TIC+Rec-RRY].

The SMB: Synthetic Multicellular Bacterium: The aim of our project is to engineer the first synthetic multicellular bacterium, the SMB. This new organism is a novel tool for the engineering of complex biological systems. It consists in two interdependent cell lines. The first, dedicated to reproduction is the germ line (red cells in the simulation below). It is able to differentiate into the second line: the soma (green cells), which is sterile and dedicated to support the germ line. The germ line is auxotroph for DAP (diaminopimelate) which is provided by the soma. There is thus an interdependency relationship. The soma, being sterile, requires the germ line for its generation, while the germ line needs the soma to complement its auxotrophy. We provide here both experimental and computational evidences that this system can work, as well as the almost complete construction of the SMB.

Towards Self-differentiated Bacterial Assembly Line: Our projects concern with the ability for bacterial cells to differentiate out of homogeneous conditions into populations with the division of labor. We aim at devices conferring host cells with the ability to form cooperating groups spontaneously and to take consecutive steps sequentially even when the genetic background and environmental inputs are identical. To break the mirror in such homogeneous condition, we need two devices respectively responsible for temporal and spatial differentiation. The implementation and application of such devices will lead to bioengineering where complex programs consisted of sequential steps (structure oriented programs) and cooperating agencies (forked instances of a single class, object and event oriented) can be embedded in a single genome. Although this "differentiation" process resemble the development of multicellular organism, we tend to use a more bioengineering style analogy: assembly line. Or maybe after some years from now, this will not be just an analogy.

Diauxie Elimination:Bacterial cells in growth phase preferentially metabolize glucose before utilizing any other sugar. This is because glucose is the most efficient sugar for growth. Only when glucose is no longer available for the cell's use does it switch to the metabolization of other sugars like lactose or xylose. When the cell switches from glucose to another sugar there is a lag period during which the cell produces the alternate, sugar-specific metabolization proteins. This delay is called diauxic lag. Our interest is in selectively eliminating the preferential processing of glucose before xylose. Xylose is a common lignocellulose sugar found in plant digests, and is therefore a ubiquitous energy source the can be used in the production of compounds such as ethanol. By eliminating xylose diauxie, bacteria such as E. coli could be made to produce ethanol much more efficiently due to the absence of diauxic lag. Our approach to eliminate xylose diauxie was to remove glucose's transcriptional control over xylose catabolism without significantly altering wildtype metabolism in other pathways. The xylose operon is under regulation of the xylose sensing protein XylR and the energy availability indicator CRP. CRP activates xylose machinery only when it is bound to cAMP, which is prevalent in the cell when glucose levels are low. CRP-cAMP along with XylR stabilizes RNA polymerase, thereby activating transcription. We took two separate approaches to altering the expressed levels of xylose machinery. One approach devised was to express a mutant of CRP in the presence of xylose that acts like CRP-cAMP even in the absence of cAMP (and therefore in the presence of glucose). By expressing this protein at higher levels than regular CRP, the expression of xylose catabolism machinery is activated. Another approach was to strengthen the promoter region in order to abolish the need for CRP-cAMP stabilization while maintaining XylR control. In effect, this makes the promoter insensitive to glucose levels but still sensitive to xylose levels. The relatively poor promoter of the xylose operator is incrementally changed to resemble the consensus promoter until XylR alone is sufficient to stabilize RNA polymerase and effect gene expression.
Bio Dosimeter: A dosimeter is a device which measures the amount of ionizing radiation an individual or object has received over time. With the renewed interest in nuclear energy in response to dwindling fossil fuel reserves, concerns over nuclear waste storage and disposal, and fear of radiological terrorism, the need for a cheap dosimeter is clear. A biological system that acts as a dosimeter would be a cheap and readily producible first response indicator that could be interpreted without training or calculation. In order to monitor radiation dose the bio-dosimeter would need to sense incoming ionizing radiation, a trait that organisms do not normally possess. However, the genetic damage that makes radiation dangerous is also readily monitored by several highly specific systems in nature. One such example of this is the Lambda Bacteriophage, which switches from the dormant lysogenic state to the active lytic state in response to genetic damage in its bacterial host. Our project uses the lambda phage genome as a radiation biosensor as it has been extensively characterized and has tight control over gene expression. The lambda bacteriophage controls switching between the lysogenic/lytic states once lysogeny has been estabilished through a bi-directional promoter (PR, PRM) with three binding sites with different affinities for repressor (Cl) and activator (Cro). The lysogen normally maintains lysogeny through a feedback loop with repressor Cl that simultaneously maintains Cl concentration within a set range and represses the Cro activator by activating the PRM promoter which expresses Cl and repressing the PR promoter which expresses Cro. However, in response to genetic damage the lambda phage exploits the host’s SOS response to cleave the repressor. As Cl concentration declines, the stronger PR promoter begins to express Cro, which competes with Cl for binding sites and represses Cl’s production. By taking the PR, PRM bidirectional promoter and placing desired response proteins such as GFP downstream of the activator Cro, the operator acts as a tightly regulated dosimeter with little chance of accidental false positives.

Development of a Biosensor Device for Detection of Several Metals Simultaneously: There exists two essential needs which enabled us to design the trimetallic probe. The need for alternative forms of oil, in fact their methods and the bioremediation of metals ions from the environment. Micrococcus luteus strain (ATCC 4698) was transformed by plasmid pUC57-S-3M, in which Fe (II), Ni (II) and V (II) were fused to the fluorescence proteins (ECFP, EYFP, and mRFP respectively and to a fluorescent protein coding region [C0061(lux I)] ligated with a signaling sequence BBa_I13207. The pUC57-S-3M was standardized for its specific sensoribility response to three metal ions and to sulfur (S), in order to detect metal contamination and/or hydrocarbons associated to these metals. Single and combination of the tree metals were used at different concentrations (0.2, 2, 50 ppm). The pUC57-S-3M was grown in the presence and absence of oxygen and/or hydrocarbon (Thiophenol). Only results from combined metals are reported. The biosensoribility was determined by the response of the pUC57-S-3M to the different concentrations of the metals. This response was measured by bioluminescence, fluorescence, DNA concentration, bacterial growth. These parameters were related to sensoribility of the pUC57-S-3M.

An RNAi-Enhanced Logic Circuit: Cancer-Specific Detection and Destruction: The Princeton University iGEM 2007 team, consisting of 10 undergraduate students, 2 high school students, and 7 instructors, envisions a paradigm shift in the way one can target cancer and destroy the resulting cancerous cells. The majority of contemporary cancer treatments utilize unspecific approaches in targeting cancer cells. Such methods do not, and indeed cannot, target a particular cancer cell type. As a result of this imprecision, these treatments tend to inflict almost as much damage upon healthy cells as cancerous ones. Such harmful side-effects often make cancer treatments such as chemotherapy or radiation therapy not only undesirable, but also less comprehensive, as their prolonged use is contraindicated. Our proposed system will resolve this unfortunate situation by enabling the detection and destruction of cancer cells in a tissue-specific manner. Our system is engineered in such a fashion as to prevent any deleterious effects on non-cancerous cells or on cells that exhibit characteristics similar to cancer cells. To this end, RNA interference (RNAi) is employed to provide an additional level of regulation and prevent pro-apoptotic genes engineered into the cells from being silenced by any epigenetic events. The added dimension of tissue-localization ensures that the system does not target any non-cancerous cell, thus, providing the most direct and effective cancer therapy. We have selected breast cancer as our initial target. Breast cancer, according to the World Health Organization (WHO) (ref), is the leading cause of cancer deaths in women worldwide. As a significant cause of cancer deaths, breast cancer serves as an appropriate cancer to target in a proof of concept implementation of our general design using the established MCF-7 cell line. In MCF-7 breast cancer cells, GATA3, a non-neuronal ectoderm cell fate regulator and a transcription factor present in and important for the maintenance of breast tissue cells, is upregulated to levels approximately thirty-two times those found in healthy cells. In our engineered system, when Gata3 is present in large quantities as is the case in MCF-7 cells, it will titrate away the designed small interfering RNA (siRNA) and allow transcription and translation of one of the pro-apoptotic factors: Bax or Bak. When present in small quantities, Bax and Bak are repressed by the binding of the designed siRNA to the engineered stretch of sequences located either 5' or 3' to the Bax or Bak gene. A helper lentiviral plasmid containing a mutated pol gene (mutant integrase) will enable the transient transfection of the entire system into the cancer cells upon lentiviral infection. The mutant integrase does not allow integration of the delivered genes into the chromosome. This transient effect will ensure that the apoptotic genes are not propagated by further cell division, thus preventing unintended proliferation of our construct.

Bacteria WarfareThis year's Purdue 2007 iGEM team will be presenting a project called "Bacteria Warfare" for this year's competition hosted by MIT in November. The team is composed of a group of undergraduates from different majors including Biomedical Engineering, Agricultural and Biological Engineering, Biochemistry, and Chemistry. Escherichia coli (E. coli) is used to conduct the microbial warfare. The design is to use two different types of transformed E. coli. One type of E. coli produces protein that triggers the death gene of its opponent (but not for itself) which expresses the production of a protein toxin. One expresses green fluorescence protein and the other expresses red fluorescence protein so that the progression of the war between types can be easily viewed and monitered. The team decided the general topic of a bacteria wafare at the end of spring 07 and started design and construction of the wafare in summer 07.

Quorumtaxis: Bacteria have evolved diverse genetic systems to sense their environment as well as respond to their surroundings in an adaptive manner. Of recent intense interest is the discovery that bacteria use pheromones that allow them to sense their own population density, a system known as quorum sensing. Another widely studied system is that of chemotaxis, in which a bacterium is able to sense and adaptively swim towards or away from a chemical agent in the environment. The Rice University iGEM team is attempting to merge these two existing natural systems (quorum sensing and chemotaxis) to produce a novel bacterial phenotype (quorumtaxis) in which the engineered cell will be able to detect and swim towards the quorum pheromone of another ‘target’ cell. This project will demonstrate the ability to produce unique complex behavior in bacteria through the modular integration of existing circuits. In addition, precise control over bacterial movement will greatly increase the complexity of systems synthetic biologists could create. The keystone of the project is the design of a novel chimeric receptor which can sense the target pheromone and then signal flagellar rotation within the cell. This project necessitates a highly interdisciplinary approach: the conceptual design and part construction requires backgrounds in biochemistry and cell biology, protein engineering elements requires experience in biomolecular engineering methods, and computational mathematics will be used to model the quorumtaxis phenotype.

Copper-based Schmitt Trigger: In our project we intend to create a copper biosensor, that would sense copper ions in growing media (based on a water sample). The advantage of biosensor over FAAS and MS techniques is, that it will sense true free copper concentration, available to biological system. It will not react to strongly ligand-bound copper and fine dispersed metallic copper. To make our sensor more robust we intend to supplement it with a threshold device, that will provide the response on a “all or nothing” basis, when copper level will exceed the critical concentration. So, our project falls into two distinct parts. The first part is a copper-responsive element, that will convert measured copper level to POPS. The second part is a threshold device with hysteresis (a Schmitt trigger), that is designed as a individual part, so that it may be used with any input.

Cyanide Biosensor: Southern Utah University's iGEM team is developing a cyanide biosensor. This project idea was inspired by the 2006 University of Edinburgh iGEM team which developed an arsenic biosensor. Like arsenic, cyanide is a toxic compound that can contaminate water. Currently, the most common ways for cyanide to come into contact with humans are through industrial wastes and through the root crop cassava. Cassava is a major part of the diet for about 300-500 million people living in the tropics and subtropics. There are currently already several methods for detecting cyanide in water. However, these methods are time consuming and require many steps. We would like to engineer a strain of bacteria that could produce a signal in response to the presence of cyanide. There are already some strains of bacteria such as Pseudomonas fluorescens PfO-1 that produce enzymes such as nitrilase/cyanide hydratase to degrade cyanide. We believe that the transcription of the genes for these enzymes may be dependent on the presence of cyanide. Therefore, we would like to modify the currently existing genes in the Pseudomonas strain so that GFP is produced in response to this toxic compound instead of the usual cyanide degrading enzymes. A bacterial strain with these capabilities may provide quicker detection of cyanide in the future.

Bio-Diode: In this project, we try to construct a biological device to imitate the function of the diode, one of the most significant parts in the electric integrate circuit. The flow of molecular signal AHL is considered as the current of electric circuit. The generator, amplifiers, blocks and detector cells are constructed with the parts provided by MIT and then are equipped in series in order to establish the cellular and molecular biological diode. Our device, which is a combination of technologies from the field of computer science, molecular biology and chemical engineering, is a breakthrough for the application of mature techniques of chemical engineering to the field of synthetic biology. Genetic RS Flip-Flop Combining the “Latch and Enable Control" conception of "Flip-flop" and synthetic biology, we designed the Genetical RS FLIP-FLOP whose output signal (Green Fluorescence) is regulated by additional input signal (the addition of IPTG). The GFP is only ignited when the input signal switches from 0 to 1 or from 1 to 0. Thus, the immediate response to emergency (input changes) enables our design to detect signal variation in a short time, which is beneficial to the control of biological systems.

YeSOil: A Yeast Sensor for real Extra Virgin Olive oil: The aim of our project is to engineer a synthetic biological network in yeast. This system will help in evaluating the quality of olive oil, one of the wordly famous product of Italy. Detection of oil quality is now possible only through expensive and bulky machines. In order to render this process easy and cheap we will modify Saccharomyces cerevisiae cells so that they will act as sensors and indicators of different oleate concentrations.

Bacterial Society: The goal of our project is to make a bacterial society that follows Pareto's principle as an ant society does. On the other word, we try to construct a bacterial system which takes "balanced differentiation". To follow Pareto’s principle found in an ant society, our model system must satisfy the three conditions shown in Fig. 1 to 4. In our model, all individual cells have the same genetic circuits but take either of stable state A (worker) or B (idler) depending on the surrounding circumstances as if they DIFFERENTIATE. They also change their states as if they DE-DIFFERENTIATE and RE-DIFFERENTIATE so that the ratio of the two cell states is well balanced.

RAP - The Recourable Auto-inhibitory Pulses. A novel expression oscillator that simulates the natural oscillations with most simplicity: Referring to the expression oscillators, the Elowitz oscillator is the first and the only typically successful one so far, in which three operons express three different transcriptional repressors, lacI, lambda C1 and tetR, respectively. The three transcriptional repressors in the Elowitz oscillators inhibit one another to generate oscillations. However, this is a relatively complex system, and protein accumulation has been observed at the single-cell scale during the oscillating cycles. While the natural oscillations usually works in pulses including a stage when the signal is reset to zero. For example, in the process of the active potential, the cardiac cycles and the muscle contraction, the neural/electric signal is triggered at the state of "zero", and then amplified until a feedback inhibiting mechanism is reinforced to blocked the amplification. At last, as the inhibition dominates, the signal falls to the state of "zero", which can be thought as the "reset". To understand and simulate the natural oscillations, we propose a method to allow the oscillator being reset to "zero" after each cycle and find that it is an still simpler method to generate oscillations. A fast-degrading DNA polymerase and a fast-degrading transcriptional repressor are engaged in this system. This oscillation works in four stages: (1)Triggering: the RNA polymerase gene is expressed by constructive promoters. (2)Amplication: the RNA transcripts the gene coding itself. (3) Inhibiting: the RNA transcripts a transcriptional repressor, which has longer degrading half-life than the RNA polymerase, that blocks the transcription of the RNA polymerase gene. (4) Resetting: the transcription of the RNA polymerase is blocked and until the transcriptional repressor degrades to a concentration below a specific level. And we found that this oscillator can be constructed in a single operon.
Celcuit: When engineering prokaryotic cell networks, one of the most difficulties is isolating signals. For one, it's necessary to get various kinds of signals to allow specific communication. In the widely-used quorum-sensing system, the quorum-sensing signal is excreted out of the cells, which in space broadcasts an identical signal to every recipient cell. In spite of the concentration gradient, that means cells with same receptor will be triggered simultaneously. And when trying to engineering a more complex system, the freedom of design is severely limited by available kinds of quorum-sensing signal producers and receptors. But if a framework allows one signal being isolated from another, it will break the those limits and allow different cells being triggered specifically. For another, space isolation is equally important in engineering. Take the isolated wire cable as an example, although all the wires utilized identical electric current as the medium for signal transport, the isolation allows identical electric current in different wires to generate different combinations of signals. While the excreted broadcasting prokaryotic signals are unable to work in this way because they share the same medium or space. Therefore, those requirements bring the problem posed in the field of bioengineering into a general topic in engineering cell-cell communications. It's necessary to establish a system with standards in cell-cell communication with expected isolation and variance in the kinds of signals.