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

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 it’s 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.