Current industry techniques for finding bacterial contamination takes anywhere from 6-72 hours. In this time span, the bacteria can escape their original confinement, spreading further across the product and diminishing yield. The goal of the Salmonella Sleuth project is to develop a tool or device that indicates the level of bacterial contamination at low cell density by using Quorum sensing.
Genetic Engineering is an incredible technology. It has the possibility to revolutionize countless areas of science from medicine to agriculture. You must also know then, of the stigma and negative connotation that too often accompanies discussion of genetic engineering. The goal of MST iGEM for the 2019 year was to do our part to combat that stigma by making the topic of genetic engineering approachable to all people.
One of the best ways to help people accept an opinion or idea that is different from what they believe is to offer some common ground. In the case of our project, we are entered the magical world of a particular boy wizard. The wet lab portion of our project focused on the designing of five different bacterial strains. Four of these strains represented the four houses of the famous wizarding school. Into each of these strains we inserted a sequence that would lead to the production of a homoserine lactone molecule. Homoserine lactones are naturally occurring signaling molecules. These molecules can signal the change in the expression of genes in other nearby cells. This is where the fifth strain would come into play. Into the fifth strain we inserted the necessary sequences to sense the four homoserine lactones from the house colonies. Each house would produce a molecule that was distinct from the other three. When the fifth strain sensed one of the homoserine lactones from a house colony it would fluoresce the color that corresponds with that house.
The idea was to have house colonies compete in a house cup. Classically, the four houses compete against each other for house points throughout the year. The house with the most points at the end of the year wins the house cup. In our work, the four house strains were plated at the periphery of a plate. The fifth, sensing strain, was placed in the center of the plate. The houses then competed for levels of fluorescence rather than house points. The winning strain was the one that produced the most fluorescence in the sensing colonies via the strength or concentration of homoserine lactone molecules. UV-Vis spectroscopy was conducted at the end of the experiment to determine the winner.
The Emerald Ash Borer is an invasive pest that is spreading quickly across the US and infecting ash trees, killing them with a 99% mortality rate. This insect has already spread across 33 states and killed hundreds of millions of ash trees in North America (see this site for more information. The goal of our current project is to insert the gene for a Cry toxin from bacillus thuringiensis into E. Coli, mutate it and find the best mutant by testing how well it attaches to the gut receptors of EAB, then move that mutant gene into the model plant, Arabidopsis thaliana.
Plant-based biosensors have immense benefits over analytical chemistry or potentiometric techniques for a variety of reasons. Plants have an inherent biological property of taking up chemicals over time and have access to a large sample of groundwater, which allows them to be more accurate than many market available testers - which often do not test for particulates or at very low concentrations. A biosensor has the added benefit of not compromising ease of use when increasing accuracy. When an appropriate indicator is selected, the average person should be able to look at a plant and immediately notice that a contaminant is present.
Our team selected Arabidopsis as the system for our biosensor and trichloroethylene, or TCE, as our first contaminant of interest. Trichloroethylene is the most common groundwater contaminant in the United States, contaminating between 9 and 34% of drinking water in the US. TCE has been classified as a carcinogen; many of its biological metabolites are highly reactive and have cancerous effects to specific organs. A plant that could be placed outside of factories possibly releasing TCE, or even a house plant to be watered by from the tap drinking water, could prove to be a powerful agent in reducing the amount of TCE consumed by humans.
Though we focused first on TCE, ideally our system could spread to a variety of groundwater or drinking water contaminants. The system is based on important developments in biosensors, namely the creation of synthetic signal transduction systems in plants and the redesign of natural periplasmic binding proteins for the detection of new ligands. So simply, a plant is exposed to contaminated water, a designed periplasmic binding protein binds that protein, a signal transduction pathway causes a reporter piece of DNA to be triggered, which in our case will lastly de-green the plant, meaning that the plant will turn almost clear when exposed to the contaminant. This will become obvious to any observer, and they can then begin to take the next necessary precautions
We investigated two approaches to defend bats from WNS while avoiding disturbing their hibernation, killing beneficial fungi, or releasing genetically-modified bacteria. We designed pathways on plasmids for the production of ocimene, a volatile organic compound, and leupeptin, a protease inhibitor, to reduce the severity of disease in different ways.
Pseudogymnoascus destructans, the fungal cause of White-Nose Syndrome responsible for the death of millions of bats in North America, grows on cave bats during their crucial hibernation. While bats are sensitive to disturbance during hibernation, those to survive until Spring are often able to recuperate after awakening to survive and eliminate their infection. Traditional responses to fungal infections, namely fungicides, indiscriminately kill beneficial and harmful fungi, while providing strong evolutionary pressure for resistance. We are instead exploring a volatile organic compound from fungistatic soils, ocimene, which has been shown to slow fungal growth. We are also investigating ways to sense P. destructans to impact the cave environment as little as possible, and compounds that may inhibit metabolism of the bats’ skin. Our hope is that by slowing growth of the fungus, we can defend bats from the disease and give them a chance to recover after hibernation.
We designed and are assembling two plasmids encoding the mevalonate pathway, along with the enzyme responsible for (E)-beta-ocimene production. We will characterize the parts by measuring in-vivo production of mevalonate and ocimene in E. coli. We will then proceed to in-vitro testing by GST-tagging ocimene synthase, purify, and prepare an assay with geranylpyrophosphate to test ocimene production at various conditions. Ultimately, we will test the inhibitory effects of our ocimene on cultures of P. destructans to assess its viability as a solution to the White-Nose epidemic.
Coal produces a significant portion of global power, but also causes pollution. Nitrogen oxides are formed and released into the atmosphere by coal burning. This depletes the ozone layer and oxidizes in air to form acid rain. The ozone layer absorbs ultraviolet radiation from the sun, shielding organisms on the surface from potentially harmful radiation. After extreme and continuing depletion, our ability to affect and harm the environment is now more apparent than ever. Human contributions to acid rain have been attributed to aquatic ecosystem destruction, soil spoiling, and structural corrosion. Nitric oxides themselves cause numerous respiratory symptoms.
We aimed to allow genetically modified organisms to remove all forms of nitrogen oxides from coal exhaust. Fixing nitrogen oxides through Cyanothece, or a similar organism, not only prevents these harmful compounds from being introduced to the atmosphere, but produces ammonia compounds as end products. Ammonia is an essential part of fertilizer, providing a path to offset costs of cleaning emissions. We standardized these nitrogen fixing genes and added them to the BioBrick Parts Registry for use by other synthetic biologists, hoping that our work is the first step to eliminating nitrogen oxide pollutants from coal flue gases.
This project aims to engineer E. coli to have an extracellular scaffold which could contain enzymes to break down the protective layer of Mycobacterium tuberculosis. In the past years there has been a sharp increase in strains of drug-resistant Mycobacterium tuberculosis. The mycolic acids contained within these strains aid in the drug-resistant ability of the disease. It also makes antibiotic treatment, the main form of treatment for Tuberculosis, very difficult. By engineering E. coli to have extracellular enzyme scaffolds using the cellusome scaffolding from Clostridium thermocellum, the mutated bacteria would be able to break down the mycolic acids. Therefore, making the recent strains of TB lose the aid in drug resistance and making TB treatment more successful.
In the bodies of people with diabetes, the ability to recognize and respond to glucose concentrations in the blood has been compromised. As a result, glucose accumulates to dangerous levels. High blood glucose concentrations can cause irreversible damage to critical organs, impairing their functionality. With parts from the iGEM registry, our team created a glucose-controlled promoter linked to a yellow fluorescence production gene in E. coli. The concentrations of glucose to which the promoter responds can be determined. Once the concentration is known, the promoter can be mutated so that it will be activated by varying concentrations of glucose and be used as a glucose sensor for people with diabetes. In the future, an insulin gene could be added to this system for use in insulin pumps, where specific glucose levels trigger insulin production in E. coli. This project aims to allow E. coli, a bacteria that requires oxygen and is easy to grow, to transport electrons out of the cell. This could have applications in powering micro and nanoelectronics.
The growing need for alternative fuel sources has sparked interest and research across many scientific and engineering disciplines. The fledgling field of microbial fuel cell development has previously relied on anaerobic metal reducing organisms such as Geobacter sulfurreduccens. This project sought to isolate genes from the electron shuttling pathway in Geobacter and transform them into the more manageable aerobic Escherichia coli. The team isolated four outer membrane cytochrome (omc) genes from Geobacter, which are vital to the extracellular transportation of electrons. The four genes - omcB, omcE, omcS and omcT - were cloned into individual plasmids. The eventual goal is to combine all four genes into one plasmid to transform into E. coli to create an aerobic, electron transporting microbial system. Applications of this project include using the modified bacteria as a power source for micro and nanoelectronics, or using it in medical research to act as a monitor inside the body. This project allows yeast cells to fluoresce in the presence of methanol, which could be used in breathalyzers or for detecting methanol in homebrew products.
The goal of this research is the manipulation of yeast cells; granting them the capability of measuring the concentration of ethanol present. This project utilizes the metabolic pathways of the yeast Pichia pastoris, which are capable of metabolizing ethanol and methanol. The enzyme alcohol oxidase (AO) is encoded in the AOXI gene and it appears to be the major enzyme involved in methanol metabolism. If both methanol and ethanol sources are present, P. pastoris will preferentially use ethanol first, which is controlled by the AOXI promoter which will allow visible detection of the expression of AOXI when fused to a green fluorescent protein gene. In supplying the yeast with ethanol and methanol simultaneously, the cells should produce the fluorescent protein after ethanol consumption; resulting in a visible color and fluorescence. The concentration of ethanol can be determined by measuring the time before fluorescence and in doing so, will make plausible the development of a breathalyzer device and additional sensor systems. This could also have applications in altering oxygen content of gasoline and detecting methanol in homebrewing products.