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. 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. 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. 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?
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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?
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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.
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Who will our project help?
 Image
from here
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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. |
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