Synthetic Life: Our Hybrid Future

Several fields of science and engineering, including synthetic biology, neuroengineering, computer science, and nanotechnology, are working toward the creation of new forms of and modifications to life. This session will explore what we can do now to create synthetic life, what has already been done, and what we might do in the future. The session will include a discussion on possible frameworks for how science and society can responsibly move forward together

Development of a Novel Enzymatic Pre-treatment For Lignocellulosic Biomass

Biofuels, fuels derived directly from living matter, present a renewable and environmentally friendly alternative to petroleum based fuels. Bioethanol produced from low input energy crops or agricultural waste is a promising fuel source because it does not interfere with the human food supply chain and the ethanol produced can be blended with gasoline. These potential sources of bioethanol are not yet commercially viable due to a polymer called lignin present in the plant’s cell wall which impedes the conversion of cellulose to glucose and the eventual fermentation of glucose to ethanol. Developing new methods for the pretreatment of lignocellulosic biomass that increase cellulose conversion and require less energy inputs would make lignocellulosic biofuels more attractive for investors. This study uses genetically engineered yeast to secrete enzymes which degrade the lignin and make it easier for cellulose to be converted to glucose. The four chosen enzymes were identified from the genomes of termites and white rot fungus. Devices producing the desired enzymes were assembled via overlap PCR amplification or Gibson Assembly. Analysis using gel electrophoresis revealed that, the Manganese Peroxidase device and the Aldo-Keto Reductase device had assembled correctly. Transforming these devices into yeast and applying it as a pretreatment has the potential to reduce costs and improve bioethanol yields

Bicistronic Design for Precise and Reliable Gene Expression

Despite having progressed extensively in the field of synthetic biology in terms of DNA synthesis, analysis and transplanting, we still cannot reliably, quantitatively measure expression of new genetic constructs. We engineered a biobrick compatible expression cassette to control transcription and translation initiation which can be reused in new genetic contexts. Previous research has shown that the Bicistronic design have much lesser variations in expression with varying genes of interest as compared to the regular monocistronic design.(Mutalik, Endy, Guimaraes, Cambray, Lam, Juul, Tran & Paull, 2013) The Bicistronic design(BCD) consists of two Shine-Dalgarno sequences in its translation element which when combined with indiscriminate gene of interests are known to reliably express within twofold of the relative target expression window. The expression levels can be controlled with the sequence of the Shine Dalgarno and promoter sequences. The four original BCDs driving Red fluorescent protein were chosen from V.Mutalik’s and D.Endy’s designs and they have very low, low, medium and high expressions. The fluorescence expression was measured using flow cytometry. The parts were made biobrick compatible using the RFC25 assembly standard. Results from the biobrick BCDs were similar as the original BCDs, which implies that scars from the restriction sites did not affect the expression levels. These parts will be submitted to partsregistry and made available to the public to be reused by other research groups

Establishing a Lung Model for Evaluation of Engineered Lung Microbiome Therapies

Benzene, a toxin and carcinogen found in air polluted by cigarette smoke, car exhaust, and industrial processes, is associated with the development of leukemia and lymphoma. Other than avoiding exposure, there is no current method to deter the effects of benzene. One potential strategy to prevent these effects is to engineer the bacteria of the human lung microbiome to degrade benzene. To evaluate this novel approach, we must verify that the bacteria remain viable within the lung microenvironment. To do so, lungs were harvested from rats and swabbed to determine the contents of the original lung microbiome. Then green fluorescent protein (GFP)-transformed E. coli were introduced to the lungs and the lungs were ventilated for five minutes before being swabbed again. The lungs were sliced with a vibratome and cultured for three days. They were analyzed under a microscope and swabbed daily to determine how the bacteria disperse upon delivery and detect changes within the lung microbiome. If results show that introduction of a new bacterial species does not significantly change the lung microbiome over time, the project can move forward to test the engineered bacteria’s viability in the lung environment and effectiveness in rescuing lung cells from benzene’s toxicity

Design of Transgenic S. cerevisiae for Enzymatic Pretreatment

Biofuels, combustible fuel produced from fermentation of agricultural biomass by microorganisms, represent one of the best possible paths forward for sustainable energy production. However, inefficiencies in biofuel production create barriers that stand in the way of their widespread adoption. One such barrier is the breakdown of lignin, a biopolymer that exists on the edge of plant cell walls which protects the sugars that are used in fermentation. Currently, lignin is broken down in energy-intensive thermal pretreatment processes. A viable alternative is the expression of lignin-degrading enzymes by synthetic microorganisms that work at standard temperatures, eliminating the need for the high-energy input of thermal pretreatment. Four lignin-degrading enzymes were selected from termites (R. flavipes) and white rot fungus(C. fioriniae PJ7) and two helper enzymes that assist in lignin degradation were selected and then optimized for expression in yeast. The genetic devices amplified were assembled using standard DNA assembly methods. Future transformation into yeast (S. cerevisiae) cells and testing of lignin-breakdown effectiveness may open up an alternative path for thermal pretreatment of biomass

Sporesat: a nanosatellite platform lab-on-a-chip system for investigating gravity threshold of fern-spore single-cell calcium ion currents

SporeSat – a lab-on-a-chip (LOC) centrifuge platform designed for integration as the payload of a small (5.5 kg), free-flying satellite – has been developed to determine the gravitational thresholds for calcium-ion channel activation of a single-cell spore from the fern Ceratopteris richardii. This fern is an important model system for gravity-directed plant-cell development during variable-gravity conditions attainable only in space flight. Calcium-ion channel activity is measured by photolithographically defined calcium ion–selective electrodes (ISEs) at opposite ends of each spore. Artificial gravity is created by rotating a disk-like platform that contains the spores in wells along with the calcium ISEs. Ground experiments reveal a maximum calcium concentration ratio at 2.2xg, between micro-ion-selective electrodes near the “top” and “bottom” ends of the spore, indicating an increasing calcium concentration at one “end” of the fern spore with respect to the other. Confocal micrographs of rhizoid formation confirm the light-induced germination. SporeSat is a spaceflight experiment that will take ~ 4 days; data will be telemetered to Earth over ~ 100 days

Measurement of Hydrogen Peroxide Influx Into Cells: Preparation For Measurement Using On-Chip Microelectrode Array

Hydrogen peroxide (H2O2) is commonly known as a toxic reactive oxidative species (ROS) for cells. Recent studies have found evidence that H2O2 is also an important cellular signalling molecule. Quantifying cellular influx of H2O2 will contribute to researchers’ understanding of the role H2O2 plays in healthy cells and cells involved in the progression of cancers and degenerative diseases. This work utilizes an assay kit and fluorescence techniques to evaluate cell lines and conditions to create a model biological system for measuring cellular H2O2 consumption. Pancreatic beta cells (MIN6), astrocytes, and glioblastoma cells (GBM43 and GBAM1) were placed in 10 μM and 20 μM H2O2 solutions for up to 5 hours. The consumption of H2O2 was measured using an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes/Invitrogen). GBAM1 cells exposed to 20 μM H2O2 displayed the fastest rate of H2O2 consumption (4.8 ± 1.2 nmol H2O2/min/106 cells), followed by GBM43 cells (1.5±0.46), astrocytes (1.1±0.24), and MIN6 cells (0.29±0.075). Additionally, the rate of consumption increased with increases in H2O2 concentration. In the future, an on-chip micro-electrode array (MEA) will be used for real-time electrochemical experiments to measure influx of H2O2 by astrocytes and GBAM1 cells with spatio-temporal resolution that the current techniques lack. The results from the electrochemical experiments will be compared to results from the assay kit to determine the ability of the MEA to accurately measure H2O2 concentration and flux. The MEA can be extended to a wide variety of cellular environments for analysis of additional real-time biological events

Cellular Model of Hydrogen Peroxide Release: In Preparation for On-Chip Sensor Measurements

Hydrogen peroxide is traditionally associated with cellular damage; however, recent studies show that low levels of H2O2 are released by cells as part of normal intercellular communication. The mechanisms of hydrogen peroxide transport, uptake and release, and biological effects are not yet well known but have important implications for cancer, stem cells, and aging. Standard H2O2 assays cannot make spatially or temporally resolved quantitative measurements at a cellular scale. Previously we developed a microelectrode array (MEA) and calibration methods for quantifying H2O2 gradients in space and time. The sensor was validated using artificial H2O2 gradients at subsecond and micrometer scale resolutions. The present study begins cellular work on H2O2 release to identify a cellular model system for MEA sensor testing. The morphology and H2O2 release from U937 human monocytes were analyzed after stimulation with ionomycin (1.2 ug/mL) and/or phorbol 12-myristate 13-acetate (PMA). Monocytes were stimulated with PMA (10 ng/mL to 150 ng/mL) for six hours. Hydrogen peroxide release was quantified over time using a traditional amplex red flurometric assay method. Mouse pancreatic beta (MIN6) cells were also tested as a negative control. Monocytes stimulated with PMA alone produced, on average, three times more H2O2 than those stimulated with ionomycin or a combination. Monocytes without ionomycin released H2O2 at 18.34 pmol/min/106 cells at 25 ng/mL of PMA. Ten, 25, and 100 ng/mL of PMA produced H2O2 significantly faster than the non-stimulated control. No significant difference was seen between PMA concentrations when ionomycin was added. These results indicate that PMA stimulated human monocytes may serve as a good model system for cellular validation of the H2O2 MEAs. In the future, biofunctionalization of the electrodes for additional molecular specificity will allow for the expansion of the method to other analytes, giving the sensor potential use in non-traditional lab environments with the ability to perform multiple assays autonomously

Lipidomic Analysis of Glioblastoma Multiforme Using Mass Spectrometry

Glioblastoma multiforme (GBM) is the most common and malignant form of primary brain tumors. It is highly invasive and current treatment options have not improved the survival rate over the past twenty years. Novel approaches and technologies from systems biology have the potential to identify biomarkers that could serve as new therapeutic targets for GBM. This study employed lipid profiling technology to investigate lipid biomarkers in ectopic and orthotopic human GBM xenograft models. Primary patient cell lines, GBM10 and GBM43, were injected into the flank and the right cerebral hemisphere of NOD/SCID mice. Tumors were harvested from the brain and flank and proteins, metabolites, and lipids extracted from each sample. Reverse phase based high performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (LC-FTMS) was used to analyze the lipid profiles of tumor samples. Statistical and clustering analyses were performed to detect differences. Over 500 lipids were identified in each tumor model and lipids with the greatest fold effect in the comparison of ectopic versus orthotopic tumor models fell predominantly into four main classes of lipids: glycosphingolipids, glycerophoshpoethanolamines, triradylglycerols, and glycerophosphoserines. Lipidomic analysis revealed differences in glycosphingolipid and triglyceride profiles when the same tumor was propagated in the flank versus the brain. These results underscore the importance of the surrounding physiological environment on tumor development and are consistent with the hypothesis that specific classes of lipids are critical for GBM tumor growth in different anatomical sites