Results

Cellular Lead Sensor: About 40% of the world does not have access to clean water. Lead is a major contaminant worldwide. In the US alone, over 1 million children ages 1 through 5 have elevated levels of lead in their blood. Current lead detection systems are expensive and require lab analysis. Home lead testing kits are inaccurate and only detect lead at very high levels. We have created a genetic circuit in E Coli that responds to lead. The promoter and lead binding protein we use are ten times more selective for lead than for other similar heavy metals. We have also incorporated a genetic amplifier into our circuit to allow us to detect fairly low concentrations of lead. Tristable Switch - The Tri-Stable Toggle Switch represents a continuation on the theme of the Toggle Switch begun by Gardner, et al to produce stable outputs in response to transient inputs. Applications such as a memory circuit and a drug delivery system are a few suggestions, but perhaps the most promising innovation lies in the design process. Our novel approach to the Tri-Stable Switch development is founded on quantitative principles, pioneering a technique to remove the guesswork from designing and debugging biological systems.

Electric Bacteria: The goal of our project is to use directed evolution to increase the current output of the electrogenic bacteria Shewanella oneidensis (affectionately referred to as Shewie in the Gardner Lab). As the name suggests, directed evolution consists of two main steps: intentionally mutating DNA and then selecting for the expression of desired traits. In the case of S. oneidensis, certain global transcription regulators in its genome have been identified as being related to the metabolic processes of the bacteria. These global transcription regulators will be mutated via error-prone PCR and transformed into S. oneidensis in hopes of altering current output. Bacteria that express greater electrogenic capability will then be selected via flow cytometry or other viable selection methods. This process of directed evolution can be repeated with previously selected S. oneidensis in order to increase the level of electrogenesis even further.

Cellular Lead Sensor: About 40% of the world does not have access to clean water. Lead is a major contaminant worldwide. In the US alone, over 1 million children ages 1 through 5 have elevated levels of lead in their blood. Current lead detection systems are expensive and require lab analysis. Home lead testing kits are inaccurate and only detect lead at very high levels. We have created a genetic circuit in E Coli that responds to lead. The promoter and lead binding protein we use are ten times more selective for lead than for other similar heavy metals. We have also incorporated a genetic amplifier into our circuit to allow us to detect fairly low concentrations of lead.
Tristable Switch - The Tri-Stable Toggle Switch represents a continuation on the theme of the Toggle Switch begun by Gardner, et al to produce stable outputs in response to transient inputs. Applications such as a memory circuit and a drug delivery system are a few suggestions, but perhaps the most promising innovation lies in the design process. Our novel approach to the Tri-Stable Switch development is founded on quantitative principles, pioneering a technique to remove the guesswork from designing and debugging biological systems.

Selection for Infection Our project attacks the following problem: can one engineer viruses to selectively kill or modify specific subpopulations of target cells, based on their RNA or protein expression profiles? This addresses an important issue in gene therapy, where viruses engineered for fine target discrimination would selectively kill only those cells over- or under-expressing specific disease or cancer associated genes. Alternatively, these viruses could be used to discriminate between strains in a bacterial co-culture, allowing strain-specific modification or lysis. This is clearly an ambitious goal, so we brainstormed a simple model of this problem suitable for undergraduates working over a summer. The bacteriophage λ is a classic, well studied virus capable of infecting E. coli, another classic model genetic sytem. We therefore seek to engineer a λ strain targeted to lyse specific subpopulations of E. coli based on their transcriptional profiles. Together, λ and E. coli provide a tractable genetic model for this larger problem, while hopefully providing lessons applicable to more ambitious, future projects.

Engineering cellular communication protocols: In order to engineer interesting and useful functions in biology, a robust and extensive range of intra- and inter-cellular signalling pathways must be available. By analogy with the Internet, where adoption of the standard TCP/IP communication protocol has enabled worldwide connectivity from supercomputers to refrigerators, such a system must be accessible to cells of different heritage and structure (different “operating systems”) with the potential for processing messages received and taking action dependent on their content. In the course of our project we identified and worked on candidates for both intracellular (PoPS Amplifier project) and intercellular (Peptide signalling project) communication pathways, and additionally made progress towards adding a new Gram-positive platform for synthetic biology to the Registry.

Shaping bacterial communities: Our iGEM project is to make a Marimo-ish gathering of bacteria. Marimo is a green spherical shaped algae, which is a popular living organism in Japan. It is considered a National Treasure because of its beautiful shape and its smoothness. Our system implementation assembles an affinity tag, communication module, size control. For years microbiologists have been using agar plates to isolate cells from each other. By spreading the diluted cells on a solid surface, we can make "colonies", dome-shaped gathering of genetically identical cells. Although convenient, this is only two-dimensional. What if we can create three dimentional (spherical) colonies with controlled/ defined size? Thus we can eliminate the plating process that everybody hates. Combined with the microfluidics devices, we might be able to pick, isolate, count, or innoculate each of the floating yet independent colonies to conduct routine works in future molecular biology Two cells are used in our system: AHL senders and receivers. Senders generates the affinity tags constitutively, while receivers generates them only when they are induced by AHL. The marimo-forming involves the following steps: (1) making the sender core by sticking with the affinity tag. (2) Insert the sender core into the receiver culture. (3) The sender core produces AHL, which make the near receivers to generate the affinity tags and GFPs. (4) The affinity tagged receiver sticks with the central sender core. This will continue until the AHL cannnot reach the marimo boundary. (5) When the AHL reached the marimo boundary, the adsorping stops, which makes a finite-sized marimo bacteria.

Light mediated behavior modulation in the fruit fly: The aim of this project is to engineer a behavior in the common fruit fly. It is well known that the fruit fly is capable of learning through reinforcement, and many experiments in classical and operant conditioning have been done to demonstrate the fly's capacity for learning and memory. By applying reward and punishment in the presence of certain neutral stimuli, the fly can make associations and learn to avoid or seek out these previously neutral stimuli. The current hypothesis in the literature is that, like humans, punishment and reward in insects are mediated by different neurotransmitters. It is believed that in insects, dopamine mediates punishment and octopamine (an invertebrate analog of norepinephrine) mediates reward. In our project we seek to further develop an existing method that allows for direct activation of these putative reward or punishment circuits by application of blue light to the intact animal. We hope to use this method to engineer defined, and even complex, behaviors in the fruit fly by using the blue light flashes to directly ‘reward’ or ‘punish’ behaving animals in real time.

A Microbial Biosensor Device Assembled with Ion Channels for Iron Detection under UV Irradiation and Different Levels of Oxygen: The Colombian-Israeli team is made up of students from different cities in Colombia and Israeli high school students. The students who are currently attending different universities pursue careers within the sciences and engineering. Each and every one of us has a different personal motivation that drives us in our daily work for this year's IGEM project. As a group, we also shar a motivation that brings us together withing our team: to put into useful practice our passion for biology, math and computer science but most of all, to be creative. We want to find new solutions and new ways of solving problems and overcoming obstacles found in science through synthetic biology. For this year's IGEM project, our team's objectives are to enhance the detection levels of the sensing device with the implementation of ion channels and to use these results as reference to develop other types of sensing devices to be used in different conditions. Biosensors are useful molecules and/or cellular tools that allow detection of the presence of different metals including iron (II/III) and other compounds, even at detection levels beyond the limits of conventional methods (Colombian IGEM. IET Synthetic Biology Journal. 2007). Last year, the Colombian IGEM team developed a microbial biosensor device for iron detection under UV irradiation using synthetic biology. This year, in association with the Israeli team, we will develop a more sensitive biosensor device, in order to detect different levels of iron, including those below that of 0.5 ppm. The device will also be tested at different levels of oxygen and UV irradiation. Plasmid isolation, preparation of competent cells and cell transformation are being currently carried out in the laboratory at the Universidad Agraria in Bogota, Colombia. New parts designed by the Colombian group as well as parts from the MIT BioBrick will be assembled, in order to construct the genetic machine. This year, sequences from both upstream and downstream will be used for our project. One of the main new features of our device will be exposed to different environmental conditions such as oxygen levels, temperatures and varied light intensities. As we carry out all of our experiments within our laboratory, we are also developing a mathematical and computational model.

Hamiltonian Path Problem: As a part of iGEM2006, a combined team from Davidson College and Missouri Western State University reconstituted a hin/hix DNA recombination mechanism which exists in nature in Salmonella as standard biobricks for use in E. coli. The purpose of the 2006 combined team was to provide a proof of concept for a bacterial computer in using this mechanism to solve a variation of The Pancake Problem from Computer Science. This task utilized both biology and mathematics students and faculty from the two institutions. For 2007, we successfully continued our collaboration and our efforts to manipulate E. coli into mathematics problem solvers as we refine our efforts with the hin/hix mechanism to explore another mathematics problem, the Hamiltonian Path Problem. This problem was the subject of a groundbreaking paper by Adleman in 1994 where a unique Hamiltonian path was found in vitro for a particular directed graph on seven nodes. We were able to use bacterial computers to solve the Hamiltonian path problem in vivo.

Bio-Powered Electricity and Light: This year's Duke iGEM team is interested in tackling problems associated with engineering bacterial systems to be easily controllable by clean, macroscopic inputs in order to create useful, macroscopic products. We became obsessed with trying to find solutions to these problems. We wanted to make bacteria we could control like a computer, with electric fields and light and heat. Not stopping there, we wanted to make bacteria that could generate their own electric fields, and their own heat, and after they'd been programmed, interacted, and formed networks, we wanted bacteria that could yield precise and complex materials. Of course, we broke this up into several smaller projects
* Electric field-activated transcription factor
* Bacterial communication with light
* Synthesis and property-control of bioplastics
* Bacterial solar fuel cell

Training E.coli: "All E.coli 's are equal, but some E.coli 's are more equal than others..." (freely adapted from "Animal Farm" by George Orwell) ... this is what George Orwell would have written, were he a synthetic biologist. In the E.coli colonies on petri dishes, all bacteria are equal; except for some special ones. Our project is about designing such special E.coli that are "more equal" than the rest: they have the ability to be trained in order to memorize and recognize their environment in the future. Their story will be presented through this wiki ...

Cling E-coli: This year Harvard's team consisted of eight undergraduate students, with backgrounds in molecular and cellular biology, biochemistry, engineering, and computer science. With the help of six faculty advisers and four teaching fellows, plus one education advisor, we devised and executed a single project with three subsections. Cling-E. Coli 1. Bacterial Targeting 2. Quorum Sensing 3. Fec Signal Transduction Our efforts were focused on engineering bacterial surface proteins to express peptides that will target bacteria to specific substrates. We then sought a biological read-out for whether the engineered bacteria interact with the substrate. For this purpose, we employed two distinct detection methods: quorum sensing and the Fec iron response system. By the end of the summer, we successfully demonstrated that bacteria expressing well-characterized tags on their surface could bind to specific substrates. Separately, we characterized the quorum system with multiple types of promoters and reporters, and we are on our way toward a combined targeting-quorum system in the near future. In addition, we are working on inserting random peptide libraries into the bacterial surface proteins; we will then screen for the ability to bind to a variety of substrates. Eventually, our project may have applicability in medical imaging and localized drug synthesis and release. Theoretically, if our project is successful, bacteria (or other microorganisms) could be targeted specifically to a certain tumor cell. At a certain density, quorum sensing may allow for the bacteria to emit a signal that could be detected by medical imaging devices. If the reporter gene encoded for drugs or vitamins, synthesis can also be evoked through enriched targeting. Furthermore, binding through a modified Fec system may allow for binding alone to transduce a signal and elicit a response in the cells.

Infector Detector: Our project tackles the ongoing problem of catheter-associated urinary tract infections. To do this, we looked at how infections develop - as biofilms - and designed a system which would be able to detect their presence. We have created a system which is capable of detecting one of the types of signalling molecules found in biofilms, AHL, and visibly report its presence by producing a fluorescent protein.

Solar Bacteria: The goal of the project is to produce bacteriochlorophyll by establishing a strong metabolic pathway in E.Coli. A generic metabolic pathway for chlorophyll synthesis in plants is given here (Generic Chlorophyll Biosynthesis). The biosynthesis of bacteriochlorophyll is similar; the part that our project focused on is shown here(Bacteriochlorophyll Biosynthesis). In order to channel the flux of carbon through the bacteriochlorophyll biosynthetic pathway, the flux through the first branch point between the native E.Coli pathway and the chlorophyll pathway must be optimized. The enzyme magnesium-chelatase is responsible for converting protoporphyrin IX to Mg-protoporphyrin IX. Because this reaction must occur to a large degree, we want to use an enzyme that has the most enhanced activity. Since all photosynthetic bacteria utilize very similar bacteriochlorophyll synthesis pathways, they all have their own versions of Mg-chelatase to perform the Mg-insertion reaction. A large part of the project is to subclone the genes for Mg-chelatse, for three photosynthetic bacteria—Rhodobacter sphaeroides (purple bacteria), Synechocystis sp. (cyanobacteria), and Heliobacillus mobilis (heliobacteria). After subcloning, the enzymes are overexpressed and their activities are measured. Besides the establishment of a strong initial input of flux through the bacteriochlorophyll photosynthesis pathway, we have also explored the latter parts of the pathway. These latter steps are crucial for catalyzing reactions that would lead to the final product—bacteriochlorophyll.

Modular two-gene oscillator Our project is a continuation of one of the projects we presented last year: a two-gene oscillator with an 'On' and 'Off' switch using LacI and LuxI. This simple relaxation oscillator could be used as a modular component for the synchronization of other complex oscillating systems such as the repressilator. Quorum-sensing coupling between cells is achieved with a diffusible autoinducer AI (AHL) made from LuxI which promotes the synthesis of LacI from pLux. As Lac accumulates, it represses pLac which controls the synthesis of LuxI.

3D light controlled "coliforming": The formation of complex scaffolds of extracellular matrix is one of the problems facing the emerging discipline of tissue engineering. One way to produce such scaffolds would be to have them secreeted by cells joined together in a three dimensional structure. We concieved the idea of using two light sources of different wavelength, impinging on a suspension of cells from differnt directions to induce the cells at their intersection to join to each other and thus create a three dimensional structure. Since we plan to implement the system in Ecoli we called it coliforming. Implementation of this concept requires two new biobricks of more general application and these are the focus of this year's submission to the Igem competition.
1. A new light receptor like the Red light receptor, but sensitive to a different wavelength. This is being developed based on work in the Spudich lab with Sensory RhodopsinII and by Alan Grossman with ComP (kinase), ComA(target) and PsfA(promotor) two component signalling system. Implementation is by dirrect sythesis of the required fusion genes, using the Geneart offer.
2. A protein generator for gas vesicles to make the suspension of cells neutrally bouyant. This is based on the work in the Cannon lab. Implemenation is by removal of restriction sites using site dirrected mutagenesis of Maura Cannon's plasmid and PCR primer based engineering of biobrick prefix and suffix.

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.

BACTOBLOOD: The necessity of inexpensive, disease-free, and universally compatible blood substitutes is undisputed. There are currently no blood substitutes approved for use in the US or the UK, and whole blood is almost always in short supply. Developing countries have the greatest need for blood transfusions, yet many lack the necessary donation and storage infrastructure and the required pool of healthy donors. To address this problem, we are developing a cost-effective red blood cell substitute constructed from engineered E. coli bacteria. Our system is designed to safely transport oxygen in the bloodstream without inducing sepsis, and to be stored for prolonged periods in a freeze-dried state.

Location, Location, Location: Directing Biology through Synthetic Assemblies and Organelles: We have taken a new and exciting approach to the iGEM challenge this year. Because UCSF is without an undergraduate program, we chose to take on 7 exceptional students (6 high school students and 1 undergraduate) from the bay area. The students' participation was conceived with help from UCSF's SEP program — the Science and Health Education Partnership between UCSF and the San Francisco Unified School District, designed to help boost student science literacy in the city. Using iGEM as a guiding framework, we created an educational program to introduce them to Synthetic Biology and experimental science. As part of this, the students worked as a team with a post-baccalaureate instructor and a group of graduate students and post-doctoral researchers to actively create an interesting and fun project. This year, we are using synthetic approaches to engineer and manipulate novel cellular microenvironments into Eukaryotic cells. It is widely appreciated that the efficiency and specificity of many cellular processes frequently depend on spatial localization, which can be achieved in different ways. For example, multiple enzymes involved in a given process frequently co-localize by binding to a common scaffold. Processes can also be localized in larger and more specialized microenvironments, such as whole organelles, through physical separation into distinct membrane compartments. Co-localization and compartmentalization allow molecular components to function in concert more effectively, and can protect the process from the external environment. Conversely, if the process involves any harmful intermediates (e.g. degradation, oxidation, etc.), compartmentalization can protect the rest of the cell. This summer, our team is creating novel microenvironments in yeast through (a) protein-scaffold interactions and (b) membrane compartmentalization. In the first project, we are reengineering the pathway output of the yeast mating response through the recruitment of exogenous pathway modulators to the Ste5 scaffold via synthetic leucine zippers. Our approach uses a new combinatorial cloning method based on type IIs restriction enzymes, allowing multi-step ligations to be consolidated into one. In a second project, we are using the same signaling pathway to engineer a new organelle in yeast. We are exploiting the observation that a unique code of modified phosphoinositide lipids is usually used to confer distinct identities to membranous compartments (i.e. organelles). Therefore, our project is based on conferring a distinct phosphoinositide composition to endosomes containing the mating receptor Ste2 by recruiting mammalian phosphoinositide phosphatases (MTMRs) or kinases (PI3K) able to produce phosphoinositides that normally do not exist in yeast. Through this, we hope to arrest trafficking of Ste2-specific endosomes to the yeast vacuole, creating a new stable organelle labeled with a unique and orthogonal molecular identity code. Creation of a new organelle could have unlimited engineering potential and a wide array of applications, such as creation of a segregated compartment dedicated to the synthesis of drugs or biofuels.

Modelling and Implementation of a Turing System: One of the most important problems in developmental biology is the understanding of how structures emerge in living systems. Several mechanisms have been proposed, depending on the observed patterns. The so called Turing patterns are based on the interaction of two effects: diffusion of some chemicals, called morphogenes, and the chemical interaction between them. It has been highly controversial whether some patterns observed in several organisms are of this type. In particular, although some systems have been identified to be of activator-inhibitor type (the most popular Turing system proposed by Gierer and Meindhart), it is still questioned if pattern formation and more generally, the appearance of functional structures can be understood by means of Turing patterns or more broadly, reaction-diffusion mechanims. One of the main goals of our project is to test different pattern formation mechanisms, not only Turing patterns, but also oscillatory and time varying structures. We propose that if the appropriate genetic construction is implanted in a colony of bacteria, the reaction-diffusion mechanism can be replaced by a genetic control system. This fact is first illustrated with the, by now classical, repressilator. Then we give two constructions of our own. The first is a modification of Elowitz system, which inclueded bot positive and inhibitory interactions. The positive ones are in fact dependent on quorum diffusible signals. In order to model these systems, we use both a stochastic pi calculus and differential equations approach. For this construction experiments are yet to be performed. For the other construction, we have two plasmids embedded in two different colonies. Each plasmid allows the bacterium to fluoresce red or green respectively. Our hypothesis is that competition between these two colonies once they are allowed to interact might function as a Turing system. For this we already have experimental results, and preliminary models.

Butanerds: We propose the use of butanol as the leading biofuel for use in internal combustion engines. Specifically, we intend to genetically engineer Escherichia coli bacteria to convert biomass into butanol for use as an energy source. This will be accomplished by introducing the genes responsible for butanol production from Clostridium acetobutylicum (i.e. endogenous butanoate pathway) into E. coli. Furthermore, we hope to increase E. coli's tolerance to solvents such as butanol.

A Genetic "Smartswitch": Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor Dr Otto H. Schmitt). The main feature of this device is to be a “smart” switch: that means a switch with memory. In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the quantitative modification of the value that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold). So far so good, however, if the switch input has a value that continually even minimally changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state. To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called Ton and Toff respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. This kind of "smart” switch, the "Schmitt Switch", is largely used in technical applications since it overcomes the instability issue; in fact, the minimal variation able to cause a change in the output must be as large as the difference between the two thresholds. Then noise becomes not so critical. The reason why the “smart” switch works that well is due to its hysteresis properties. Our genetic circuit aims to reproduce this fundamental property of Schmitt Trigger.

Developing A Genetic Printer: The project we selected was to design and build a biomechanical printer; composed of a two dimensional plotter equipped with a red laser, software to translate computer images into instructions for the plotter, and E. coli cells engineered to respond to the laser light. Bacteria are spread in a solid lawn on the plate, or mixed in the media before pouring the plate. The response triggered by this biological circuit will produce beta agarase, an enzyme which degrades the agar polymer that the cells rest on. The printer can then be used to "draw" high resolution images on the bacteria with the laser. The bacteria will then dissolve the agar where the laser was shone. This results in Bacterial Lithography, where the dissolved agar forms a picture. Cool eh. We also chose a second project to include in our entry to the competition this year. That is an in Silico Biobrick Evolution system. The purpose of this project is to design a system that will accept user entered parametres and use them to search through the registry database. Using the given parametres the system will try to construct circuts (a series of biobricks) that will produce the desired product. More information on this project can be found in our evoGEM sections. Our team investigated a number of potential projects before selecting our light sensing printer. This section offers a brief outline of some of the ideas we considered.

Division PoPper: The Division PoPper is a signal generator device that produces an output of PoPS as a function of bacterial cell division. In simple terms, it is a device that generates a "pulse" of PoPS signal each time a cell undertakes division. Downstream of the device may be a counter device, quantifiable protein production or some other function of choice. The system may for example perform pre-determined actions such as programmed cell death after a set number of cell divisions or being used to calculate the division frequency. The device relies on dif recombinase sites to flip a DNA segment at each cell division. With this project we hope to further analyse cell division and recombinase mechanisms since bacterial cell division is still relatively poorly understood. We plan to construct and ligate the bricks required for a first proof of concept experiments. We model the device using various approaches (deterministic and stochastic modelling) in order to guide lab work and analyse lab data. Modelling is also used to simulate the composition of our device with a counter device. In presenting our work, we highlight with comments and discussion the application of Synthetic Biology approaches at each work phase.
Self-flavouring Yogurt: We chose to produce 'self-flavouring' yoghurt, as this project enables us to genetically manipulate a range of gram positive bacteria required for yoghurt formation (see yoghurt production, below). There are several advantages for using genetically engineered gram positive bacteria, over the traditionally used E. coli. * Some Gram positive organisms (eg Lactobacillus, Lactococcus) are food grade, and therefore may be ingested. Lactobacillus are alsp common and harmless (indeed, beneficial) components of the human gut flora. Potentially this could enable their use in a range of products from medicines to milk shakes or yoghurt to deliver beneficial molecules to the body. Of course, there are also regulatory issues related to consumption of genetically modified organisms, but these will be more easily addressed in a known-harmless host with a long history of safe food use. * Several gram positive bacteria, such as Bacillus spp. are able to form hardy endspores, which are extremely tolerant of heat, drying and other stresses, and can survive for centuries. These do not require refrigeration and can be transported and used in very hot, cold or developing countries cheaply and with ease. * Gram positive organisms, such as the lactic acid bacteria, are much more efficient at secreting proteins and other molecules into their surrounding media than E. coli; most strains of E. coli lack the Main Terminal Branch of the General Secretory Pathway and secrete only a few proteins using specific mechanisms such as ABC transporters (eg, for hemolysin) and Type 3 Secretion Systems (in pathogenic strains).

Integrated Sensor-Executor Proteins and Molecular Switches: Our goal is to design integrated molecular sensing and executing devices based on modular protein engineering. These integrated devices can then easily be used for the construction of complex systems. We fuse sensing proteins, which provide nano-mechanical movements or dimerization upon an external signal, to executing proteins, which depend in their activity on the nano-mechanical change in the sensing part. To elucidate the possibilities of such a system we used the calcium-ion sensor Calmodulin and the light sensor system PhyA-Fhy1. To test execution we used the split enzymes DHFR or beta-lactamase or the fluorescent proteins CFP and YFP, which can form a FRET pair. Sensors and executors were geneticlly fused and tesetd in E. coli for activity. So far we could demonstrate Ca2+ dependent growth of E. coli with the DHFR[1]-Calmodulin-DHFR[2] construct. Biobrick compatible strategy for fusion proteins:The present BioBrick prefix and suffix rules are not compatible with modular protein design. Thus, we propose an extension of the present standard for fusion proteins in which two restriction sites are added in frame adjacent to the coding sequence.

ElectrEcoBlu: Our project aimed to design and construct a completely novel type of self-powering electrochemical biosensor, called ElectrEcoBlu. The novelty lies in the fact that the output signal is an electrochemical mediator which enables electrical current to be generated in a microbial fuel cell. ElectrEcoBlu functions as a biosensor for a range of important and widespread environmental organic pollutants which stimulate the biosensor to produce its own electrical power output. The system has the potential to be used for self-powered long term in situ and online monitoring with an electrical readout. Our approach exploited a range of state-of-the art modelling techniques to support the design and construction of this novel synthetic biological system. This was facilitated by the entire team - biologists and modellers - working in an integrated laboratory environment.

Bio-prospector: The goal of our project is to create a ‘bio-prospector;’ a microorganism capable of moving up a concentration gradient for a particular small molecule (theophylline), and activating either a metabolic or signaling pathway. We hope that once this system is developed, it will be easy to modify it to be specific to many different small molecules. To achieve this, we will use a riboswitch (Topp and Gallivan, 2007) to control the levels of CheZ, a protein involved in motility.

Virotrap: A Synthetic Biology Approach Against HIV: We have devised a synthetic system of antiviral defense against the HIV-1 infection that is not sensitive to viral mutations, because it is based on viral functions. Two essential viral functions have been successfully implemented to activate the cellular defense – viral attachment to cells through a pair of surface receptors and processing of viral proteins by its own protease. Binding of virus to human T-cells causes formation of CD4-CCR5 heterodimers, which in our system reconstitutes the split ubiquitin. This protease cleaves-off the membrane-anchored T7 RNA polymerase from the membrane, directing it into the nucleus. T7 RNA polymerase provides the amplification of the signal and causes transcription of versatile effector genes, coding either for antiviral proteins or for caspase, which leads the infected cell into apoptosis thereby preventing further spread of viral infection. The same viral function was successfully utilized in the implementation of the split TEV protease system. The second implementation of this idea was to utilize the activity of HIV-protease, which is required for viral maturation and cleaves a specific amino acid sequence. This target sequence was engineered between the membrane anchor and T7 RNA polymerase. T7 RNA polymerase released from the membrane subsequently activates the defense similar to that described with the split protein system. All three systems work in human cells. We have prepared and tested many different constructs, contributing more than 70 new BioBricks and successfully demonstrated activation of response gene by infection of mammalian cell cultures with HIV-1 pseudovirus.

Divide by two circuit: The DBT circuit is a single-input toggle switch with the green and red fluorescent proteins (GFP and RFP for short) to monitor product levels. If the system is at a steady state, with high concentrations of GFP and low RFP levels, adding arabinose will cause a switch in the protein levels. As a result, another steady state will be reached in which RFP is at a high concentration and there are small GFP levels. Adding arabinose again causes a switch back to the original steady state of the system (GFP at high levels, RFP at low ones). The crucial elements of the circuit are the two AND-gates, which require two specific molecules in order to activate transcription of the gene driven by the specific promoter. The specific mechanics of the DBT circuit are as follows: If we start at a state where the levels of GFP, glnK, cI are high and the levels of nifA, nifH, RFP, and the sigma-54 factor are low, by adding arabinose, we increase the levels of sigma-54. The high levels of sigma-54 and glnK causes the lacI gene to be transcribed by “activating” the glnKp promoter. lacI inhibits further production of GFP, glnK, and cI. At the same time, because of the low levels of nifH, cI will not be transcribed as much as lacI. By the time the arabinose is used up, the levels of GFP, glnG, and cI will have decreased substantially and the levels of nifA, lacI, and RFP will have increased. Similar behavior to the one described above will occur if arabinose is added again. Nested landing pad: The goal of this project is to develop tools to aid synthetic biologists in inserting genes and other DNA sequences onto the E. coli chromosome. While a few “simple” landing pads are already available for placement of DNA onto the chromosome, our project seeks to develop a general strategy for constructing landing pads that are exceptionally easy to place onto the chromosome and have advanced features. Among these advanced features are limiting noise from external promoters, allowing simplified phenotype screening and genetic analysis, allowing BioBrick-compatability, and allowing sequential insertion of DNA modules into the same chromosomal locus, where they will form a set of nested sequences.

A biological timer: We plan to make a timer from a synthetic genetic circuit. Past iGEM designs of clocks have been successful, so we decided to build on this idea. Our idea is to fashion a genetically modified network within E. coli cells which is regulated by and input signal, and emits an output, or “off” signal after a given amount of time. The input for the circuit will be a specific amount of arabinose and the output will be fluorescence. The bacteria, which will continually synthesize GFP, will be fed an input signal of arabinose sugar. During consumption of the sugar, the GFP production will be repressed. This hiatus in the fluorescence signifies a timed period. Upon complete consumption of the sugar, the bacteria will once again actively generate GFP, signaling that time is up and the timer is off. The amount of arabinose fed to the cells will determine the amount of time it takes for them to fluoresce. This timer device will be created as an individual “machine,” independent of a larger network or device. We are curious to discover if construction of such a device is possible before we try to integrate it into a larger system. Additionally, we plan to add at least one new BioBrick part to the parts registry for future use. In June we began the process of choosing and replicating four different BioBricks necessary for our device.
A biological breathalyzer: The purpose of this research is to use recombinant technology to culture yeast cells capable of determining the concentration of ethanol and using these cells to construct a breathalyzer. A complex pathway for the metabolism of methanol exists within some species of the Pichia genus. Alcohol oxidase (AO) appears to be the first and major enzyme produced in this metabolic pathway (1). Transcribed from its gene (AOX1), AO converts methanol to formaldehyde within the yeast’s peroxisome (1). A metabolic pathway for the utilization of ethanol is also present within the yeast. However, if both ethanol and methanol is present, the yeast will utilize the ethanol before consuming the methanol (2). Consequently, the AOX1 gene will not be expressed to produce the AO enzyme until the ethanol has been consumed. Fusing the AOX1 gene promoter with the DNA sequence of a fluorescent protein will allow the expression from the AOX1 promoter to be detected. In supplying the yeast cells with ethanol and methanol simultaneously, the cells will produce the fluorescent protein once the ethanol is utilized; resulting in a fluorescent light. The concentration of ethanol can be determined by measuring the time before a fluorescent light is emitted. Determining the concentration of ethanol present will also make plausible the construction of a breathalyzer device to determine the blood alcohol level in an individual. Constructing a biological breathalyzer will require various interdisciplinary skills to accomplish. Biological techniques are required to construct, amplify, and introduce the vector containing the fused DNA sequences into the yeast cells. Knowledge of the biochemical reactions is needed to understand the ethanol and methanol metabolic processes to manipulate the yeast cells to produce a visual response to ethanol. An engineering approach is necessary to design and construct the device so as to introduce methanol once the ethanol has been supplied by the individual, as well as determining and displaying the blood alcohol level.

An Extensible Logic Circuit in Bacteria: Our Artificial Bio-Logic Circuit is composed of "logic gates" and "wires" like Digital Electronic Circuits. Though we have been enjoying the advantages of ultra-large-scale electronic circuits in modern life, we still cannot implement a somewhat small-scale circuit in vivo with several levels of gates. Our project is to provide a new method for building up a fully extensible bio-logic circuit in bacteria. A small fragment of DNA containing cis-acting elements, favored for its small scale and potential to implement complex logic computation in vivo, can be systematically built up and act as a gate. Meanwhile, artificial repressors with highly-specific DNA-recognition regions are able to transmit signals without mutual interference, just as enameled wires. In this way, a circuit can be constructed regardless of the number of logic gates and the layout of the wires. A demonstration system has also been assembled to show the practicality of this method. Just like Digital Electronic Circuits in early days, it is simple and ugly. Nevertheless, how will it appear in future?

Bacterial Neural Network: Our project aims to build a bacterial (E. coli) neural network composed of two cell types, where filter cells (type A) modulate input to reporter cells (type B). The first cell type is stimulated with red light of a specific intensity and duration, and will turn blue in proportion to that "pulse" of light. Populations of type A will be physically mounted above those of type B, acting as a light filter. Type B cells are receptive to the same wavelength of light, and will fluoresce in proportion to the amount of light they receive. Neural networks are unique in that their ability to signal both forward (type A influencing type B) and backward (type B influencing type A) results in the ability to learn. Training sessions can be performed with predefined inputs and outputs, and repeated iterations will increase the probability that giving the network an input will produce the desired output. Our neural network training functionality will be implemented through cell-cell signalling from type B cells to type A cells, adjusting the strength of the light filter. Depending on the training strategy used, our neural network can learn to function as either an AND or an OR gate. Essentially, our neural network will be able to sum a number of inputs and provide a proportionate output. Once training is completed with a few inputs, we should be able to provide novel inputs to the network and produce appropriate responses. This is a step towards demonstrating fuzzy logic (as opposed to traditional digital logic) in genetic circuits.

Light controlled biobutanol synthesis: The novel microbial metabolism developed by the 2007 Virginia Genetically Engineered Machine (VGEM) Team for the production of butanol from cellulose and light is composed of four main systems that comprise the pathway- cellulose metabolism, light metabolism, butanol biosynthesis, and butanol tolerance- have extended E. coli K-12’s natural central metabolic pathway. In this way, a valuable chemical product such as butanol biofuel can be biologically manufactured using inexpensive feedstock material. The cellulose and light metabolizing systems may one day serve as engines in future system designs to drive forward the biosynthesis of chemical products in microorganisms.

An Adding Circuit for Bacterial Computation: The goal of this project is to design a basic device for computing. Our idea was to reproduce a circuit element called a half adder with DNA, which takes in two 1-bit inputs, adds them, and outputs a sum and a carry. Our device responds to two inputs: red light and the chemical tetracycline. The input sensors control a set of genetic switches in order to carry out the computation and fluoresces green, red, or neither, depending on the outcome. Half adders are used as building blocks for full adders, which perform calculations similar to long addition but in binary. They are also an essential component in a device called the Arithmetic Logic Unit (ALU), a fundamental building block for the central processing unit in a modern computer. ALUs perform simple and complex operations such as bitwise logical operations and mathematical operations. The constructs for the half adder were built in parallel as well as the testing constructs. A future extension to this project would be to create a full adder. More information on each stage of the project is available below.

Artificial Transcription Factor: Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells. Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors. We decided to use zinc fingers for the DNA-binding domain. A zinc finger is made of about 30 amino acids and is stabilized by a single zinc ion to form a certain protein structure (eg. beta-beta-alpha). It can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by using zinc finger to recognize and bind to a specific DNA sequences, and then recruite other molecular machinery that will activate or repress the gene. Our ATF project has focused on characterizing DNA binding specificity and cooperativity effects

Harvesting Cellulose and Light to Power Butanol Biosynthesis: The potential and flexibility of the comparator idea have been of crescent importance to choose it as our project. This idea fits perfectly in the iGEM project philosophy of modularity as it could be a very useful piece for more sofisticated projects in future. This was the main reason to select it as our project. Because of the simplicity in the structure of the project it will be possible to carry out wet lab experiments in the limited time period of the competition. Furthermore, as a consequence of its versatility, we can increase the complexity of our project as much as we want if we see that we have time to do it. Starting with a simple, robust circuit we think that it could be of interest to the scientific community who have been working on sensing bacterias. The basic structure of the project can be explained as follows. Initially if we have an equal amount of external parameters such as any chemical compound, the expression level of both the repressor will be equal and which in turn will results in equal level of GFP and RFP (or other fluorescence proteins), which are essentially the reporters for the external variables or parameters. Then, one of the two sensor devices (let's say sensor 1) starts to have more strengh and, so, its repressor 1 and its reporter 1 are transcripted in some more concentration. This repressor 1 inhibits the promoter from the other construction (let's say 2), so we have less repressor 2 and reporter 2 from the construction 2. Finally, we have an increase on the first fluorescence and a decrease on the second one: that way we have compared two sensing strenghts and amplified the stronger one. Applications include promoter calibration, a proportional controller, discrete level detector, ADC signal convert, and a three-fluorescence output.

The Comparator: The potential and flexibility of the comparator idea have been of crescent importance to choose it as our project. This idea fits perfectly in the iGEM project philosophy of modularity as it could be a very useful piece for more sofisticated projects in future. This was the main reason to select it as our project. Because of the simplicity in the structure of the project it will be possible to carry out wet lab experiments in the limited time period of the competition. Furthermore, as a consequence of its versatility, we can increase the complexity of our project as much as we want if we see that we have time to do it. Starting with a simple, robust circuit we think that it could be of interest to the scientific community who have been working on sensing bacterias. The basic structure of the project can be explained as follows. Initially if we have an equal amount of external parameters such as any chemical compound, the expression level of both the repressor will be equal and which in turn will results in equal level of GFP and RFP (or other fluorescence proteins), which are essentially the reporters for the external variables or parameters. Then, one of the two sensor devices (let's say sensor 1) starts to have more strengh and, so, its repressor 1 and its reporter 1 are transcripted in some more concentration. This repressor 1 inhibits the promoter from the other construction (let's say 2), so we have less repressor 2 and reporter 2 from the construction 2. Finally, we have an increase on the first fluorescence and a decrease on the second one: that way we have compared two sensing strenghts and amplified the stronger one. Applications include promoter calibration, a proportional controller, discrete level detector, ADC signal convert, and a three-fluorescence output.

Engineering an Epidemic: Using modern air travel, it is possible to travel around the world, often several times, within the time it takes for a viral infection to show symptoms. This complicates the early detection of epidemics, making it even more important to predict whether an infection will become an epidemic as early as possible. If an infection is "treated" on a worldwide scale early enough, an epidemic could be prevented. Since transportation and communications technology has changed so rapidly in recent years, research needs to be done to find new ways of combating epidemics. With this problem in mind, we set out to revolutionize an approach to modeling epidemics. We wanted our model to be testable, which is vital to verify its accuracy. Therefore, we would not be reliant on historical records like many epidemiological approaches do. These records can be imprecise or inaccurate, and we knew we could get more accurate data from our testable system. We also wanted to make more use of stochastic modeling, using equations that take random variations into account. The event of an infection either becoming an epidemic or dying out in its early stages is a highly random event influenced greatly by small fluctuations among individuals. The model we built is multi-scale, specifically covering the growth of the host, and the spread of the phage within an isolated subpopulation. We also envisioned a third layer, the spread of the phage between artificially connected subpopulations. Integrating these levels of modeling to produce varying high-level population epidemic behavior by changing low-level parameters is one of the key aspects of our project. Building an understanding of high-level dynamics from the very lowest level of organization will allow us to more accurately predict the epidemiological outcome. An important part of modeling is testing the model to see if it works in real life. To do this, we needed a model host and pathogen system. We decided to use E. coli and bacteriophage λ (lambda phage) since as a system they are well-studied and cheap and easy to work with. Lambda phage is also interesting because it can either immediately kill the host cell or it can insert its DNA and lie dormant. The decision to stay dormant (lysogeny) or to kill immediately (lysis) added a degree of flexibility to our model that allowed us to check the model robustness for slightly different phage. As part of the project we designed a reporter plasmid to generate florescent proteins and indicate the virus’ decision. This plasmid, along with the use of a phage modified to be florescent, we hoped, would allow us to determine how the population would act and verify our models.