Difference between revisions of "Team:ICT-Mumbai/Design"
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| − | <p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;">Ammonia, an obnoxious molecule is perceivable even at concentrations as small as 0.04 ppm (Ref. 1). Therefore to eliminate this odor, | + | <p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;">Ammonia, an obnoxious molecule, is perceivable by the human nose even at concentrations as small as 0.04 ppm (Ref. 1). Therefore to eliminate this odor, we planned to conjugate ammonia to an organic substrate, rendering it odorless in the process. To our surprise, the humble bacterium <i>Escherichia coli</i> has already figured how to do this!</p> |
| − | <p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;">Under nitrogen starvation, <i>E. coli</i> assimilates ammonium by conjugating it to glutamate | + | <p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;">Under nitrogen starvation, <i>E. coli</i> assimilates ammonium by conjugating it to glutamate to from glutamine, an odorless molecule. Our project revolves around harnessing this reaction and making it more efficient.</p> |
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| − | <p style="height:70 px;font-family:'Lato'; font-size:20px; colour:lightgrey;"> | + | <p style="height:70 px;font-family:'Lato'; font-size:20px; colour:lightgrey;">When ammonia concentrations are lower than 50 μM, passive diffusion can no longer drive ammonia transport into the cell (Ref. 2). In such situations, a specific ammonium transporter carries ammonium across the membrane in unionised form. This ammonium is then conjugated to glutamate in a reaction catalyzed by glutamine synthetase (GS) to form glutamine. The amino group of glutamine is then transferred to alpha-ketoglutarate to form two molecules of glutamate, a reaction catalyzed by glutamate synthase (also called GOGAT). 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. We have tried to make this a cycle that can be activated on demand.</p> |
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| − | <p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;">Native <i>E. coli</i> glutamine synthetase is produced by expression of <i>glnA</i> gene | + | <p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;">Native <i>E. coli</i> glutamine synthetase is produced by expression of <i>glnA</i> gene that is downstream of a promoter that is responsive to nitrogen starvation. We decided to construct a plasmid that will enable overexpression of <i>glnA</i> under an inducible promoter. This will allow high rate of ammonium assimilation, whenever needed.</p> |
| − | <p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;">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 <i>et al.</i>, within first 15-30 seconds of adding | + | <p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;">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 <i>et al.</i>, within first 15-30 seconds of adding 10 mM ammonium to <i>E. coli</i> 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 <i>et al.</i> as an ATP-conserving process. To overcome this problem, we will be utilizing proteorhodopsin, a light-driven proton pump that will create an artificial proton gradient and help in ATP production via ATP synthase (Ref. 4).</p> |
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| − | <p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;"> | + | <p style="height:70 px; font-family:'Lato'; font-size:20px; colour:lightgrey;">We introduced an element in our design to keep track of the amount of ammonia assimilated. Indigoidene, a blue-colored compound, is formed by non-ribosomal peptide synthesis (NRPS) from glutamine in a single step reaction catalyzed by the product of the <i>bspA</i> gene. A colored compound that is produced in proportion to the amount of ammonia assimilated would indicate when cells would have to be replenished.</p> |
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Revision as of 07:00, 1 November 2017
Ammonia, an obnoxious molecule, is perceivable by the human nose even at concentrations as small as 0.04 ppm (Ref. 1). Therefore to eliminate this odor, we planned to conjugate ammonia to an organic substrate, rendering it odorless in the process. To our surprise, the humble bacterium Escherichia coli has already figured how to do this!
Under nitrogen starvation, E. coli assimilates ammonium by conjugating it to glutamate to from glutamine, an odorless molecule. Our project revolves around harnessing this reaction and making it more efficient.
Ammonia assimilation in E. coli
When ammonia concentrations are lower than 50 μM, passive diffusion can no longer drive ammonia transport into the cell (Ref. 2). In such situations, a specific ammonium transporter carries ammonium across the membrane in unionised form. This ammonium is then conjugated to glutamate in a reaction catalyzed by glutamine synthetase (GS) to form glutamine. The amino group of glutamine is then transferred to alpha-ketoglutarate to form two molecules of glutamate, a reaction catalyzed by glutamate synthase (also called GOGAT). 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. We have tried to make this a cycle that can be activated on demand.
Engineering ammonia assimilation
Native E. coli glutamine synthetase is produced by expression of glnA gene that is downstream of a promoter that is responsive to nitrogen starvation. 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 10 mM 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 utilizing proteorhodopsin, a light-driven proton pump that will create an artificial proton gradient and help in ATP production via ATP synthase (Ref. 4).
Real-world applicability
We introduced an element in our design to keep track of the amount of ammonia assimilated. Indigoidene, a blue-colored compound, is formed by non-ribosomal peptide synthesis (NRPS) from glutamine in a single step reaction catalyzed by the product of the bspA gene. A colored compound that is produced in proportion to the amount of ammonia assimilated would indicate when cells would have to be replenished.
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.
