Nitrogen Fixation

Goals:

Isolate the genes necessary for functional nitrogen fixation and assemble them into a single part, rather than the natural form of multiple gene clusters scattered throughout the organism.
Insert this part into our recipient organism and have it display evidence of nitrogen fixation.
Examine if our synthetic, nitrogen-fixing organism can actually aid in plant growth (by a significant amount) in model plants.

How Are We Meeting These Goals:

As detailed in the Notebook section, our initial strategy of focusing on a single temperature for our PCR experimentation did not work out well (only for nif fragments 2 and 3). It was then that we decided to employ a linear gradient scheme to increase the temperature across 12 different plots in the thermocycler (the temperature difference between plots 1 and 2 is the greatest, while the temperature difference between plots 11 and 12 is the smallest). While this turned out to be a successful method for the majority of the fragments, nif fragment 1 still failed to show up on agarose gels. After optimizing and fixing our primers, we were finally able to get nif fragment 1 to show up on a gel.

Anf Cluster (Expected 7220 bp)

vnf Fragment 1(Expected 6683 bp)

vnf Fragment 2 (expected 5646 bp)

nif Fragment 1(Expected 10286 bp)

nif Fragments 3 (Expected 3341 bp and 7420 bp)

nif Fragment 4 (Expected 9736 bp)

Once we had these PCR products, we performed Gibson assemblies in order to get our desired product (the single part that contains all genes necessary for nitrogen fixation). Once we obtained this product we transformed it into NEB E. coli 10 Beta (optimized for large inserts such as ours), and allowed it to grow overnight on LB tetracycline plates. After successful growth, we selected for E. coli with the insert on Nitrogen-Free, Nitrogen-Deficient, and Nitrogen-Enriched media under anaerobic conditions with only leucine supplementation. We then attempted to confirm the presence of the insert in colonies isolated from these plates via colony PCR.

E. coli 10 Beta with Gibson product, growing on tetracycline plate

We are currently selecting for E.coli 10 Beta with the correct Gibson product in an anaerobic chamber. If we’re able to see growth in an oxygen-free environment, on nitrogen-free/deficient media, then it will confirm that our recipient organism is in fact able to fix atmospheric nitrogen into bioavailable ammonia (and in enough quantity to support metabolism). We also tested R.palustris CGA010 and P.protegens PF5 on these media anaerobically

Note: Media has some inherent particulate. All PF5 plates (- ctrl) showed no growth and all Palustris (+ ctrl) plates showed robust growth.

For future experimentation, we plan to inoculate Arabidopsis thaliana seedlings (our model plant) with our recipient organism, and evaluate whether our organism can supply ammonia to the plant and if it is in high enough levels to support plant growth (instead of just bacterial growth). Additionally, if this organism can support plant growth, we will evaluate whether it does a comparable job to commercial fertilizers.

Team:OhioState/Results

Nitrogen Fixation

Goals:

How Are We Meeting These Goals: