Team:Wageningen UR/Wetlab
Wetlab
Cattlelyst is built on three pillars: Methane oxidation, Ammonia removal, and Safety. Each pillar encompasses several wetlab projects. Below all seven projects are listed.
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Synthetic methane oxidation in Escherichia coli
Soluble and particulate methane monooxygenase, and methanol dehydrogenase were expressed in C1 auxotrophic E. coli strains, connecting this C1-molecule to an efficient conversion pathway from methane biomass and carbon dioxide.
Synthetic nitrification in Pseudomonas putida
Nitrification genes originating from autotrophic nitrifier Nitrosomonas europaea were expressed in P. putida. This allows P. putida to convert ammonia to hydroxylamine and subsequently nitrite, the first steps in the conversion of ammonia to dinitrogen in our biofilter.
Synthetic denitrification in Pseudomonas putida
Denitrification genes originating from several native denitrifiers were expressed in P. putida. This allows P. putida to convert nitrate or nitrite to dinitrogen gas via the intermediates nitric oxide and nitrous oxide. These are the final steps in the conversion of ammonia to dinitrogen in our biofilter.
Limiting nitrous oxide production in Pseudomonas putida
Steady nitrogen conversions are ensured by redirecting the electron flux towards the denitrification machinery in P. putida. This was realized by establishing a denitrification machinery or downregulating the native ability of P. putida to respire with oxygen, in parallel.
Methane-dependent kill switch in Escherichia coli
This kill switch couples methane concentration to a toxin-antitoxin system in E. coli. Should the bacterium escape the biofilter, the methane concentration would drop, leading to toxin production and cell death.
Proximity-dependent kill switch in a microbial coculture
This kill switch kills P. putida and Escherichia coli upon escape from the biofilter. Outside the biofilter, they lose the close proximity to each other, activating the kill switch.
Bacterial co-dependency
A co-dependency was established between E. coli and P. putida by creating a cross-feeding community. The cross-feeding is based on amino acid exchange/auxotrophy and a carbon-source dependency.