Motivation

The growth of human civilization has been closely linked to fertilizer production for over a century. Before the development of the Haber-Bosch process in 1910, nitrogenous fertilizers were difficult to manufacture on an industrial scale. In the following century, agricultural production exploded and global population soared from 2 to 7 billion people. However, such demand for nitrate fertilizers has taken a toll on the environment. Nitrates are easily washed from farmland into tributaries and larger bodies of water, where plants and microbes are unable to consume them quickly enough. Instead, algae and other organisms proliferate rapidly in the excess of nutrients and then die off, leaving massive, oxygen-depleted 'dead zones.' In the graph below, you can see how aqueous nitrate levels in the Mississippi River have not only risen in recent years¹, but are also correlated with the size of the dead zone where it empties in the Gulf of Mexico².

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Nitrates can also leach into groundwater and aquifers. When used by people for drinking, nitrate-contaminated water can be toxic³. It is associated with “blue baby syndrome,” a potentially lethal condition.

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The Nitrogen Project was initiated to fund research that investigates ways to mitigate nitrate use in agriculture and slow the impact of runoff.

Our Project:

A key area of research for the Nitrogen Project has been to get nitrogenase, the enzyme that fixes gaseous nitrogen and converts it to usable nitrates, into non-diazotrophs, which are bacteria that cannot fix their own nitrogen. Past efforts have been made to transform the nif gene cluster, which codes for nitrogenase, into E. coli, but no conclusive results were found.

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In order to solve the nitrate problem, we propose creating a “Super Cell,” a cell that overproduces ATP and reduced electron donors in order to provide nitrogenase with a suitable environment for maximized activity. We overexpressed genes involved in ATP and electron donor production and created constructs that ultimately increased the intracellular concentrations of these co-factors. We also conducted a proof of concept experiment to show these co-factors being utilized by the cell.

Additionally, we improved the characterization a Heat Shock Promoter (HSP) BioBrick that is supposedly activated at high temperatures but had no previous data on the registry. Our characterization experiments, which were repeated by Vilnius iGEM, showed that this HSP was only slightly activated by high temperatures (42°C) and its activity greatly decreased over time.

To start learning about our project, find out how we designed our Super Cells