Project Overview
Our project is currently the first step in a much larger
endeavor.
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We
are attempting to take the nif cluster from a
cyanobacteria and get it to function in E. coli while
simultaneously attempting to create a transcriptional regulation system
that turns off in the light and turns on in the dark.
In doing such, we
hope to create a system for nitrogen fixation that operates exclusively
in the absence of light in preparation for transformation into a
photosynthetic system. After we come to a greater understanding of how
the system works and perfect it, we can move on to working in a more
complex organism, such as a cyanobacteria like Synecosystis spp.
6803.
The end goal is to create plants that can fix their own nitrogen
by moving from the cyanobacteria into the chloroplast of the plant.
Endosymbiotic theory postulates that cyanobacteria are the ancestors to
chloroplasts, so this is the natural progression. |
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This summer, we were successful in transforming E. coli with the nif cluster from Cyanothece 51142, and ran an
Acetylene Reduction Assay in order to test for the strains ability to
fix nitrogen. For more information, please consult our
Nitrogenase page.
This summer, we were also successful in cloning a light regulated
repressor system in order to turn OFF transcription of genes in the
presence of light. For more information on our methods and background,
please visit the Light
Regulation page.
Be sure to check out our
Collaboration and Parts pages as well! |
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Organism
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Ease
of Engineering
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Photosynthetic
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Crop
Plant
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E. coli
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✓✓
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✕
|
✕
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S. 6803
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✓ |
✓ |
✕
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Chloroplast
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✕
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✓ |
✓ |
Our teams project goals were to:
• Determine the optimal conditions for culturing E. coli strains containing the Cyanothece sp. 51142 nif cluster
• Select the best strains for further testing
• Create a light repressed gene regulatory mechanism
• Compare fold change of light induction with new
hybrid promoter
Figure above: Engineered
strains of E. coli being
flushed with argon gas to create anaerobic conditions
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Through
our experiments, we concluded that:
• Of the five E. coli strains tested, JM109 and
WM1788 showed strongest nitrogenase activity.
• The linear relationship between nitrogen fixation
activity and time matches that seen in nature.
• Optimal conditions: glucose as carbon-source,
glutamate as nitrogen-source, LB as inoculating media, minimal M9 as
testing media for GC assay, anaerobic environment at 30 °C
for overnight preparation before acetylene reduction assay.
• Troubleshoot a faulty reporter mechanism
• Created a hybrid promoter
• Ran light experiments that showed discernable fold
change in on and off states with appropriate amounts of aTc.
In future, we intend to:
• Alter conditions to increase activity in JM109 and
WM1788
• Determine a minimal nif cluster
• Directly check and optimize light sensitive
promoters
• Adjust the leakiness of the light sensor system to
not need aTc
• Swap out the reporter protein with the nif cluster
to get both systems working in conjunction.
• Transition into cyanobacteria by transferring the
genes with the nif cluster back into Synechocystis S. 6803. |
References
The pictures used above were taken
from the following sources:
1.
Synechocystis
2.
E. coli
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