Missouri Miners Championship Poster

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
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
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
• hmp - forms nitrate from nitric oxide
• nosZ - converts nitrous oxide to nitrogen gas
Utilize pre-existing parts:
• nrfA (Bba_K113001): reduces nitrite to
• 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.
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
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,
Transform parts into cyanobacteria and
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