Clearing the Air: Nitrogen Oxide Fixation for the Remediation of Coal Flue Gases Missouri University of Science and Technology Key project researchers: Alie Abele, Kira Buckowing, Kelsey Crossen, Blythe Ferriere, Levi Palmer Project contributors: Hannah Frye, Kent Gorday, Joshua Heath, Aaron Jankelow, Hannah Kim, Matthew Lindner, Keith Loveless, Emily Puleo, Jordan Sanders, Caleb Trecazzi Introduction Abstract Results Coal is a key source of energy in the modern world, yet its emissions pollute the air and its toll on human health is believed to account for more than 13,000 premature deaths, Figure 1: Missouri S&T coal plant 20,000 heart attacks, and 1.6 million lost workdays in the U.S. each year.1 Environmentally friendly filtration of industrial pollutants is a critical issue facing the developed world. Carbon dioxide, sulfur oxides, and nitrogen oxides are the common pollutants emitted from coal plants. Some microorganisms are known to take these pollutants from the air and convert them into an inert substance. Several genes in E. coli K-12 and P. aeruginosa PAO1 can be used to fulfill these goals and create ammonia fertilizer as a useful by-product. By utilizing synthetic biology to create an organism with these desired genes, we can use bioreactors to remove pollutants in a environmentally healthy manner. The goal of this project is to modify the genome of Cyanothece 51142, a cyanobacterial species, to allow it to fix a variety of nitrogen oxide compounds that are major pollutants present in coal flue emissions. The Missouri S&T iGEM Team plans to clone and standardize genes from Pseudomonas aeruginosa and Escherichia coli that will allow Cyanothece 51142 to take up and convert nitrogen oxides into ammonium, a major component of fertilizer. This could lead to a method to reduce polluting emissions while producing fertilizer to offset the costs of scrubbing the emissions. • hmp successfully ligated into standard pSB1C3 backbone, sequenced for gene verification, and submitted to the iGEM registry (see Figure 4 and 5). • Could not successfully amplify nosZ, PCR product consistently incorrect • norV isolated, but did not successfully ligate to test viability Project Goals Create BioBrick parts: • norV - reduces nitric oxide to nitrous oxide • hmp - forms nitrate from nitric oxide • nosZ - converts nitrous oxide to nitrogen gas Utilize pre-existing parts: • nrfA (Bba_K113001): reduces nitrite to ammonia2 • norCB (Bba_K896005): reduces nitric oxide to nitrous oxide3 Incorporate these parts into cyanobacteria, a photosynthetic microorganism already capable of fixing carbon dioxide. Figure 4: hmp plasmid Figure 5: Digested hmp/pSB1C3 ligation product Part Submitted Figure 2: Simplified Project Schematic Joe Microbe investigates our project’s coal plant filtration system using genetically modified cyanobacteria for nitrogen fixation with hmp, norV, nosZ, nrfA2, and norCB3. The products and reactants of the reactions performed by each gene are shown (for simplicity, other reaction components were omitted). Nitrogen gas and ammonia are produced as inert by-products. Nitrous oxide is also produced and could potentially be extracted for “laughing gas”, but this process would need to be further explored. Cyanobacteria naturally converts carbon dioxide to oxygen through photosynthesis. Methodology Outline: 1. Isolate genes from E. coli K-12 and P. aeruginosa PAO1 2. Functionalize parts with promoter, RBS, and terminator 3. Submit parts to BioBrick registry Making the Parts (See Figure 3): 1. Design primers for nosZ from P. aeruginosa PAO1 and hmp and norV from E. coli K-12 a. Primers add iGEM prefixes and suffixes 2. Amplify desired sequences 3. Conduct site-directed mutagenesis for norV (and eventually nosZ) a. Remove internal PstI site 4. Insert into desired vector References: 1. Schneider, C., and Jonathan Banks. 2010. The Toll From Coal: An Updated Assessment of Death and Disease from America’s Dirtiest Energy Source. Clean Air Task Force, September. 2. University of Kent iGEM Team – 2013 3. National Yang Ming University – Taipei iGEM Team – 2012 4. Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., ... & Poole, R. K. (2002). NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp. The EMBO journal, 21(13), 3235-3244. We would like to thank… Part BBa_K1370000 is the isolated hmp gene from Escherichia coli, and produces a protein, dihydropteridine reductase, which catalyzes the reduction of nitric oxide to nitrate.4 The synthesis of its products are elevated in response to nitrite or nitric oxide through transcriptional regulation. Future Work • • • • • • Figure 3: Project Steps Outline Troubleshoot norV ligation Sequence nosZ to determine what PCR product is Remove internal site for nosZ Functionalize by adding promoter, RBS, terminator Transform parts into cyanobacteria and express Test potential flue emission filtration systems for design implementation • Advisors: Dr. Katie Shannon and Dr. David Westenberg • Donor Fred Kielhorn • Missouri S&T cDNA Resource Center, SDELC, Student Council, and the departments of Biological Sciences, Chemistry, and Chemical Engineering