Team:WashU StLouis


WashU IGEM received a Silver Medal for participating in the Giant Jamboree! Congratulations to everyone involved~

Additionally, we are now recruiting for 2015. Please follow this link for the application!

Engineering E. coli to Fix Nitrogen and Regulating Transcription with Light

Why are we doing this project?

Synthetic biology is an exciting area of research that aims to genetically improve organisms to make them more efficient and hopefully more useful to us as well. The human population in 1950 was 2.5 billion, yet it is predicted to surpass 9 billion by 2050 [1]. Even with population growth slowing, increasing life spans and standards of living will soon tax our natural resources. One of the most concerning is our food supply. The agriculture industry needs a revolution in order to keep up with our expected growth rates. Currently about 80% of chemically fixated nitrogen is used as agricultural fertilizers, the majority in developed lands [1]. Intracellular nitrogen fixation in crops could help to sustain the burgeoning world population, especially in areas with less fertile soil without taxing the planet’s waterways. The exponential increase in nitrogen fertilizer has led to more runoff into rivers and oceans. Fertilizers then provide nutrition for algal blooms that result in hypoxia and form oceanic dead zones. These dead zones lead to the death of marine species and have potentially large economic consequences.

The ramifications of nitrogen fertilizer runoff can be averted by genetically engineering plant crops to fix their own nitrogen. Some cyanobacteria fix nitrogen for nutritional needs, while most organisms can only acquire it from the food it consumes. Synthetic biology allows us to transfer this ability to fix nitrogen to a heterologous host that has many genetic tools, Escherichia coli, so that we can learn how to give single cell organisms, and eventually chloroplasts the ability to create their own nitrogen fertilizer.

Diazotrophic (organisms that fix nitrogen) cyanobacteria such Nostoc Punctiforme or Anabaena use heterocysts (specialized nitrogen fixing cells) to create a mini-anaerobic environment to aid nitrogen fixation. However, Cyanothece 51142, a non-heterocyst, fixes nitrogen in the same cell as photosynthesis by relying on a circadian metabolic process, when there is less oxygen byproduct from photosynthesis. This process is both fascinating and necessary since the key enzyme in nitrogen fixation, nitrogenase, is poisoned by oxygen. Our goal this summer is to engineer the regulation of the proteins necessary for nitrogen fixation so that they are highly repressed when activated by broad spectrum light (such as the sun), and are highly active when there is no light around, mimicking the cycle where photosynthesis occurs during the day and nitrogen fixation occurs at night.

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How does our project work?

WashU project Our team worked this summer on two projects towards a common purpose.
Richard and Caroline worked in the Pakrasi lab under Dr. Pakrasi, with Andrew Ng, Bert Berla, and Deng Liu as advisors on the getting nitrogen fixation working in E. coli.
Benjamin and Jeffrey worked in the Moon lab under Dr. Moon, with Cheryl Immethun as an advisor, and created a repressor system to turn off transcription of a reporter protein in the light.

For more information, please visit our project tab.

What did we accomplish?

There are two key components of our project: 

Richard and Caroline have been working in the Pakrasi lab, and have been using genes from cyanobacteria to get nitrogen fixation working in E. coli . They are testing nitrogen fixation by running acetylene reduction assays and designing experiments to test the optimal criteria (ie. E. coli strains, temperature, pH, nitrogen source) to get maximum results. For more info, visit the nitrogenase tab.

Ben and Jeffrey have been working in the Moon lab, and have been busy cloning plasmids to create a system that represses and expresses a fluorescent protein with the presence and absence of light and running experiments to test the induction levels compared to various positive and negative controls. For more info, visit the light regulation tab.


Who will our project help?

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Our project is the first step of a much larger, much more complex endeavor. Nitrogen overabundance and nitrogen depletion are simultaneously big stumbling blocks in modern agriculture. The solution to both of these problems would be to endow plants themselves with the ability to fix nitrogen so that they could autonomously supply their own nitrogen for proteins, DNA, etc. We are taking the first step towards this ambitious goal by studying how the genes for nitrogen fixation from cyanobacteria work in different environments and constructing an artificial transcriptional system. We are currently working in E. coli because it is easy to engineer, but the next step would be to move into a cyanobacteria more closely related to chloroplasts. We hope that by making these initial steps that we may be helping to pave the way for future research that may put an end to world hunger.