Project Design

We started with our brainstorming and project design stage where we thought through various directions we could take our idea, and then we moved into the experimental design stage where we outlined the things we wanted to accomplish over our project's duration.

Brainstorming Stage

Using synthetic biology to reduce methane emissions from cattle was a well-accepted project idea from the beginning. The team’s first hurdle was figuring out how to go about pursuing this goal. One of the earliest ideas was to inhibit methyl coenzyme M reductase, one of the final enzymes in methanogenesis. The far most appealing inhibitor was 3-nitrooxypropanol with one study finding it decreased methane production by 71.5% (Romero-Pérez et al., 2016). However, we could not pursue this method because a biosynthetic pathway for 3-NOP is not known and the compound falls under patent protection for the specific use of decreasing methane production in ruminants, Patent No. WO 2012084629 A1.

A short-lived idea was to design E. coli to be a methanotroph, having methane be its one and only carbon source to counteract methanogenesis. Unfortunately, information on methanotrophy inside bovine rumina is lacking (Attwood & McSweeney, 2008). Dr. Niu, one of our advisors, along with Dr. Fernando from UNL’s Animal Science Department recommended that we look elsewhere as this would be a difficult undertaking.

Our last pitch incorporates the idea to redirect the flow of hydrogen away from methanogens (Satyanagalakshmi et al., 2015), since the removal of hydrogen is essential for cofactors to be reoxidised and for ruminal fermentation to be maintained (Attwood & McSweeney, 2008). Our team researched this route and initially found sulfate-reducing bacteria to be a prime candidate for reducing methanogenesis. However, hydrogen sulfide is a product of sulfate reduction and can cause serious neurological damage to organisms (Drewnoski et al., 2011).

As a team, we decided to pursue the two most plausible options for our project. The first solution we decided on was biosynthesis of bromoform to inhibit methanogenesis (Kinley & Fredeen, 2014). The second solution involves aiding the facilitation of nitrite reduction to ammonia (Yang et al., 2016). Additionally, the specific enzymes we selected function at the pH (6-7) of ruminal fluid and even more so under anaerobic conditions.

Experimental Stage

We successfully cloned and transformed E.coli to carry the gene for the enzymes nitrite reductase and vanadium dependent bromoperoxidase. Before moving on to the next step we made sure to fully sequence our composite parts. Upon sequencing we found that there were no mutations so we decided to began the characterization of our various parts. To characterize nitrite reductase we performed the Nessler’s test. More information on the Nessler's test can be found on our Experiments page. To characterize the bromoperoxidase we used the monochlorodimedone assay which is commonly used to determine the rate at which the enzyme brominates hydrocarbons. More detailed information on these steps can be found in the lab notebook and results sections. Although we made plans to go further with the experimental design, at this point we ran out of time.

Shoulda, Coulda, Woulda

Unfortunately we were not able to carry out our full experimental design.

1. The next step that should be taken is to see if our E. coli can grow in filtered ruminal fluid.
2. If it could survive in anaerobic conditions similar to the rumen of a cow (anaerobic, same temp, same pH) while growing in filtered ruminal fluid then the assays should be reperformed for each enzyme while the bacteria is in filtered ruminal fluid.
3. Next the assays need to be repeated while the bacteria is growing in unfiltered ruminal fluid.
4. If the bacteria can grow in the unfiltered ruminal fluid then we would test if the methanogens within the ruminal fluid were still able to produce as much methane.
5. If the last few steps were successful, the kill switch would be ligated into our plasmid. If the kill switch works then begin to recharacterize the parts to make sure that they still work with the addition of the kill switch.
6. After this create the delivery system for feeding the E. coli to cattle. In the spirit of iGEM we wished to continue applications of the work done by the Oxford 2016 iGEM team making agarose beads. This would apply well to delivering bacteria to cattle because the bacteria will remain encapsulated inside the beads during shipment yet when it is inside the cow the protein will still be able to diffuse out. The bacteria and its required substrates would be put inside the agarose beads and added as a top dress onto the basal diet of the cattle.
7. After this we would have used our connections at UNL to visit the Mead Research Center and test our E. coli on cows there to see the results in vivo.

Works Cited:

• Attwood, G., and McSweeney, C. (2008) Methanogen genomics to discover targets for methane mitigation technologies and options for alternative H2 utilisation in the rumen. Australian Journal of Experimental Agriculture 48, 28–37.
• Drewnoski, M., Beitz, D C., Loy, D. D., Hansen, S. L., and Ensley, S. M. (2011) "Factors Affecting Ruminal Hydrogen Sulfide Concentration of Cattle," Animal Industry Report: AS 657, ASL R2587.
• Duval, S., and Kindermann, M. (2012, June 28) Use of nitrooxy organic molecules in feed for reducing methane emission in ruminants, and/or to improve ruminant performance. Patent No. WO 2012084629 A1
• Kinley, R. D., and Fredeen, A. H. (2014) In vitro evaluation of feeding North Atlantic storm toss seaweeds on ruminal digestion. Journal of Applied Phycology 27, 2387–2393.
• Romero-Pérez, A., Okine, E., Guan, L., Duval, S. M., Kindermann, M., and Beauchemin, K. A. (2016) Effects of 3-nitrooxypropanol and monensin on methane production using a forage-based diet in Rusitec fermenters. Animal Feed Science and Technology 220, 67–72.
• Satyanagalakshmi, K., Sridhar, G. T., and Sirohi, S. K. (2015) An overview of the role of rumen methanogens in methane emission and its reduction strategies. African Journal of Biotechnology 14, 1427–1438.
• Yang, C., Rooke, J. A., Cabeza, I., and Wallace, R. J. (2016) Nitrate and Inhibition of Ruminal Methanogenesis: Microbial Ecology, Obstacles, and Opportunities for Lowering Methane Emissions from Ruminant Livestock. Frontiers in Microbiology 7.