Growing and Harvesting Nitrogenase Proteins from Azotobacter Vinelandii Kara Dunne-Dombrink, Faith Heffernan, Dr. Akif Tezcan

The Department of Chemistry & Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, California

To study the worth of the electronic interaction between constituent proteins FeP and MoFeP, in the enzyme Nitrogenase, I am growing and harvesting different types of bacteria: A wild type bacteria, Azotobacter vinelandii which is naturally found and can make Nitrogenase easily, the mutant βK400E, which changes a positive ion on the MoFeP to become negative (thus causing a weaker connection between the FeP and the MoFeP), and finally L127∆, which takes away a part of the FeP, causing the FeP to become stuck in place to the MoFeP.

Abstract

Conclusions

Future Directions Growing the Mutants

Background • Ammonia

• Needed by countless organisms • Pharmaceuticals • One of the highest-produced chemicals in the world • Can be made by Nitrogen fixation (slow):

• Nitrogenase • Only enzyme that can turn dinitrogen from the atmosphere into ammonia • Within the enzyme itself, the Fe protein (FeP) transfers 1 electron to the connected

MoFe protein (MoFeP) with each association

• Cracking cells open to find proteins • Purifying proteins • Testing:

• Changing variables: pH, salt concentration, etc. • More Mutants

Acknowledgments

N2+ 8 e- + 16 ATP + 8 H+ 2 NH3+ 16 ADP + 16 Pi + H2

FeP

MoFeP

Wild Type

Specific Primers

• Wild Type: [N+] • Grown in fermenter • Measure amount of ethylene to know when to harvest bacteria

βK400E: [N “slow”] • Activity of mutant is not as efficient as WT • Could cause cells to grow slower

L127∆: [N -]: • FeP stuck in place on MoFeP • We hypothesize that the cell will not be able to produce ammonium from the

nonviable Nitrogenase

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K400E OD and Ethylene Production vs Time

Ampicillin Resistance

By growing and harvesting different types of cells such as the Wild Type, βK400E mutant, and L127∆ mutant, I learned about the different effects each strain has on the proteins produced. I saw that the K400E mutant produced about the same amount of Ethylene, but at a quicker rate than the Wild Type. The L127∆’s OD seemingly took longer to grow and gave us a smaller amount of protein because the cells make defective Nitrogenase that does not allow them to produce ammonia. We tried to harvest the L127∆ at the point when the cells were still making Nitrogenase and had the MoFeP and FeP that we want to crack out of the cells.

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This work was supported by the NIH (5R01GM099813-01) and the Department of Chemistry and Biochemistry at UCSD. Thank you to all the people in the Tezcan Lab, especially Cole Carter and Cedric Owens.

← MoFeP

← FeP

Ladder K400E

Ladder WT

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L127∆ OD vs Time

← FeP

← MoFeP

Growing the Wild Type

Growth Conditions in Fermenter Burke’s medium (BM) : 0.2% sucrose 0.9 mM CaCl2 1.67 mM MgSO4 0.035 mM FeSO4 0.002 mM Na2Mo2O4 10 mM Na3PO4 (pH 7.4) 10 mM NH4Cl

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WT Growth pH vs Time

(Correlates to L127 ∆ growth)

C2H2 + 2e- + 2H+ C2H4