Project Overview

Our project is currently the first step in a much larger endeavor.

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.

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!

Organism	Ease of Engineering	Photosynthetic	Crop Plant
E. coli	✓✓	✕	✕
S. 6803	✓	✓	✕
Chloroplast	✕	✓	✓

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

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

3. Corn

Team:WashU StLouis/Project

From 2014.igem.org

Project Overview

References

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