The world needs fixed nitrogen

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2 The world needs fixed nitrogen World population (millions) % World population Average fertilizer input (kg ha ¹ yr ¹) Erisman, JW, et al. Nature, Year 2

3 Problems with Haber Bosch Energy-intensive Creates greenhouse gases Fertilizer run-off 3

4 An alternative to Haber Bosch Cyanothece sp photosynthesizes and fixes nitrogen Separates them temporally Nitrogenase enzyme need reference Bandyopadhyay, et al. Nature,

5 From Cyanothece to plants? Cyanothece related to chloroplasts Can we engineer nitrogen-fixing plants? Diagram courtesy of Wikimedia.org 5

6 The nitrogen project Images courtesy of cfb.unh.edu, landcareresearch.com, geneticliteracyproject.org, edenbrothers.com 6

7 Engineering nitrogen-fixing E. coli...faster characterization of the Cyanothece nitrogenase, which would be used to... Expressing a minimal Cyanothece nif cluster in E. coli would lead to... Images courtesy of columbiariverkeeper.org, wikipedia, and ucdavis.edu...engineer a better diazotrophic Synechosystis 7

8 Nitrogen fixation is not easy for E. coli The nitrogenase reaction: 16 H2O + 16 ATP + 1 N2 + 8 Reduced Flavodoxin 16 ADP + 16 Phosphate + 2 NH4 + 4 H+ + 1 H2 Cyanothece sp. ATCC s nif cluster is 35 contiguous genes Cyanothece promoters, transcription factors, and RBSs may be incompatible with E. coli 8

9 Project overview 9

10 Refining the nif cluster 2014 igem Team Native nif cluster - 35 genes Regulatory Barriers Unnecessary genes Uncharacterized genes 2015 igem Team Our designed nif cluster - 14 genes Inducible Expression Optimized synthetic RBS A Minimal Set of Genes 10

11 The minimal nif cluster 2 plasmids for simpler cloning Operons organized by expression level Structural Iron Molybdenum Synthesis Iron Sulfur Synthesis Nitrogenase Stabilization Inducible promoter (ptrc) Inducible promoter (arabad) Strong RBS Weak RBS 11

12 CRISPR/dCas9 allows for targeted knockdowns Blocks gene transcription Knockdown genes on minimal cluster 3 sgrnas per gene/promoter Qi, et al. Cell,

13 Overexpression plasmids 14 plasmids of nif genes Inducible promoter 13

14 Results Sequence-confirmed minimal nif cluster plasmids arabad-cyse2usvwzhesbkan-rep101 Sequence-confirmed CRISPR/dCas9 plasmids H1 H3 D1 D2 K1 K2 K3 ara1 ara3 E1 E2 E3 N2 N3 S1 S2 W1 W2 Z1 Z2 hesb1 hesb2 hesb3 Trc1 Trc2 B2 V2 Restriction-digestconfirmed overexpression plasmids cce_0551 cce_0552 cce_0555 cce_0556 cce_0562 cce_0566 cce_0567 hesa nifb nifen nifv nifx 14

15 Status of wet lab work Complete minimal nif plasmids Testing First complete Second on its way CRISPR/dCas9 targeted knockouts 27 complete 21 on their way Acetylene reduction assay protocol developed by 2014 member Caroline Focht Overexpression plasmids 12 complete 2 on their way 15

16 Introduction to modeling Genome-scale model (GSM): set of metabolic reactions identified for a given organism Gene-Protein-Reaction (GPR) relationships Reaction directionality Scaled biomass equation Flux Balance Analysis (FBA): paired with GSMs to estimate metabolic flux through organism (Orth et al. PNAS, 2010) 16

17 Main objectives Goal: Optimize nitrogen fixing E. coli using computational modeling Task 1: Identify media supplements to increase ATP production and growth for N2 fixing cells Task 2 : Perform in silico single and double gene knockouts Task 3: Identify flux redistributions between diazotrophic and non-diazotrophic E. coli 17

18 Media supplementation Supplemented glucose in existing media with additional substrate equivalent to 60 Identified 15 metabolites as having: Largest increase in ATP production per increase in max biomass Larger increases in ATP production than additional glucose 18

19 In silico gene knockouts Iteratively performing FBA Double gene knockouts: computationally intensive Want to couple metabolite production to biomass No coupling found between flavodoxin reduction and biomass Single Gene Knockouts Double Gene Knockouts (50% of total) No effect on biomass Some effect on biomass Lethal

20 Changes in pyruvate metabolism Flux variability analysis: cells allocate more flux through pyruvate synthase (POR5) under N2 fixing conditions POR5 produces reduced flavodoxin In silico pyruvate dehydrogenase knockout (PDH) = increased flux through POR5 PDH KO leads to pyruvate buildup in cell2 Recommended in vivo PDH knockout, combined with POR5 overexpression (adapted from Voet, Voet) 2. U.S. National Library of Medicine. Result Filters. National Center for Biotechnology Information. 20

21 Human practices Agriculture-focused panel presentation open to the WashU community Discussion included safety, regulation, ecological effects, labeling, potential to solve problems in the world Team and attendees got a space to have questions answered, learn, and think critically about important issues Hope that it will spark further discussion on campus 21

22 Part characterization collaboration with Vanderbilt igem Ran induction experiments to determine validity of the part Part K RFPyy did not work in either strain 22

23 Registry characterization Part is TetR-pTet system that expresses RFP when induced First transformation produced cells that were red in color K in DH10B 23

24 BBa_K characterization Performed a second transformation 24

25 BBa_K characterization Performed a third induction experiment Results highlight the discrepancy in the two transformations 25

26 Added 12 new composite RBS parts to the registry On each BioBrick: Constitutive ptet promoter RBS mrfp Two terminators RBSs used for genes in minimized nif cluster Differing levels of expression 26

27 Special thanks to our sponsors... The Focht, Bourg, and Heeney/Toomey Families NSF-MCB Award #

28 Special thanks to our mentors... Tae Sook Moon Fuzhong Zhang Costas Maranas (Left to right) Carlos Barba, Cheryl Immethun, Yi Xiao, Andrea Balassy, Thomas Mueller, Young Je Lee, Ray Henson, Caroline Focht 28

29 Learn more about our team and project WashU igem Visit our poster: Hall C, No