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Clearing the Air: Using Synthetic Biology for Remediation of Coal Flue Gases

Abstract

The goal of this project is to modify the genome of Cyanothece 51142, a cyanobacteria 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 uptake 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.

Background/Inspiration

Fossil fuels are currently the most prevalent source of energy in the modern world, accounting for over 80% of our total energy consumption as of 2010 according to the REN21’s (Renewable Energy Policy Network for the 21st century) 2012 renewables global status report. Although work is being done on alternative energy sources, it does not seem like fossil fuels will go away in the foreseeable future. In order to utilize fossil fuels for their energy, they must be combusted in a power plant. This process releases flue gases, which contain several major pollutants such as carbon dioxide, sulfur oxides, and nitrogen oxides. These pollutants have negative effects such as toxicity and contributing to acid rain. However, there exist certain microorganisms that can take these pollutants from the air and convert them into something less harmful or something useful. In some cases they can even create useful chemicals such as ammonia from these pollutants. Therefore, by utilizing synthetic biology to create an organism that can deal with several of the biggest pollutants, we can use bioreactors to remove these pollutants in a cheap and efficient manner.

Project Goals

Create BioBrick parts:

• norV - reduces nitric oxide to nitrate
• hmp - catalyzes formation of nitrate from nitric oxide
• nosZ - converts nitrate into nitrogen gas

Utilize pre-existing parts:

• nrfA (Bba_K113001): reduces nitrite to ammonia
• norCB (Bba_K896005): reduces nitric oxide

Incorporate these parts into cyanobacteria for use in a bioreactor for filtering flue gases. Cyanobacteria are photosynthetic, so they are also capable of fixing carbon dioxide, another common pollutant.

Outline of Project Steps

1. Isolate desired genes from E. coli K-12 and P. aeruginosa
2. Functionalize parts with promoter, RBS, and terminator
3. Submit parts to BioBrick registry
4. Insert parts into Cyanothece 51142
5. Incorporate into bioreactor to filter flue gases

Making the Parts

1. Design primers for nosZ from P. aeruginosa PAO1 and hmp and norV from E. coli K-12
1. Primers add iGEM prefixes (EcoRI and XbaI) and suffixes (SpeI and PstI)
2. Amplify desired sequences
1. Issues with nosZ – PCR product is incorrect size
3. Site-directed mutagenesis for norV (and eventually nosZ)
1. To remove internal PstI site
4. Insert into desired vector
1. Tried to put promoter and RBS into pSB1C3, then ligate parts – ligation issues
2. Hmp successfully ligated into pSB1C3
3. norV isolated, but did not successfully ligate to test viability

Part Submitted to the Registry

Part BBa_K1370000: hmp in pSB1C3
Size of hmp: 1191 base pairs.

Next Steps

• Troubleshoot norV ligation
• Sequence nosZ to determine what PCR product is
• Internal site removal for nosZ
• Functionalize by adding promoter, RBS, terminator
• Transform parts into cyanobacteria and express