Team:ICT-Mumbai/Design
DESIGN
Ammonia, an obnoxious molecule is perceivable even at concentrations as small as 0.04 ppm (Ref. 1). Therefore to eliminate this odor, our main aim was to conjugate ammonia to an organic substrate, rendering it odorless in the process. To our surprise, the E. coli seemed to already worked it out!
Under nitrogen starvation, E. coli assimilates ammonium by conjugating it to glutamate , converting it into glutamine, an odorless molecule. Our project revolves around harnessing this reaction and making it more efficient.
Ammonia assimilation in E. coli
In presence of ammonia concentrations lower than 50mM, passive diffusion can no longer drive ammonia transport into the cell (Ref. 2). At such times, a specific ammonium transporter carries ammonium across the membrane in unionised form. This ammonium is then conjugated to glutamate in a reaction catalysed by glutamine synthetase to form glutamine. The amino group of glutamine is then transferred to alpha-ketoglutarate to form two molecules of glutamate. Glutamate and glutamine share a very interesting relationship wherein glutamine replenishes the cellular glutamate pool which in turn initiates the GS-GOGAT cycle again. However efficient this cycle may seem, it is activated only when cells are under stress along with other issues. We have tried to assimilate all such parameters in our design.
Engineering ammonia assimilation
Native E. coli glutamine synthetase is produced by expression of glnA gene placed downstream of a promoter that responds only to nitrogen starved conditions. We decided to construct a plasmid that will enable overexpression of glnA under an inducible promoter. This will allow high rate of ammonium assimilation, whenever needed.
One critical factor we came across while doing so was the continuous amount of ATP that would be required. Glutamine synthetase requires ATP for its function. According to Schutt et al., within first 15-30 seconds of adding 10mM ammonium to E. coli cells, there is a 20 fold increase in glutamine levels but a 90% decrease in ATP levels (Ref. 3). This is followed by inactivation of glnA which has been described by Schutt et al. as an ATP-conserving process. To overcome this problem we will be utilising proteorhodopsin, a light-driven proton pump that will create an artificial proton gradient that supplements the native one and helps in ATP production (Ref. 4).
Real-world applicability
Even though our bacteria could take care of ammonia odor in washrooms, their cleanliness will still remain a doubt since ammonia odor is usually caused due to stagnating urine. We found a need for introducing an element in our design that is produced proportional to the rate of ammonia assimilation.
Indigoidene, a blue dye is formed by non-ribosomal peptide synthesis (NRPS) from glutamine in a single step reaction by bspA gene. The blue color will act as an indicator of two things: 1) Need to clean the washroom 2) Need to replace the culture with fresh cells.
References
- https://hazmap.nlm.nih.gov/category-details?table=copytblagents&id=291
- Javelle, Arnaud, et al. "In vivo functional characterization of the Escherichia coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase." Biochemical Journal 390.1 (2005): 215-222.
- Schutt, Hermann, and Helmut Holzer. "Biological function of the ammonia‐induced inactivation of glutamine synthetase in Escherichia coli." The FEBS Journal 26.1 (1972): 68-72.
- Walter, Jessica M., et al. "Light-powering Escherichia coli with proteorhodopsin." Proceedings of the National Academy of Sciences 104.7 (2007): 2408-2412.
