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The world needs fixed nitrogen Average fertilizer input (kg ha ⁻ ¹ yr ⁻ ¹) World population (millions) % World population Erisman, JW, et al. Nature , 2008. Year 2
Problems with Haber Bosch ● Energy-intensive ● Creates greenhouse gases ● Fertilizer run-off 3
An alternative to Haber Bosch ● Cyanothece sp. 51142 photosynthesizes and fixes nitrogen ○ Separates them temporally ● Nitrogenase enzyme need reference Bandyopadhyay, et al. Nature , 2010. 4
From Cyanothece to plants? ● Cyanothece related to chloroplasts ● Can we engineer nitrogen-fixing plants? Diagram courtesy of Wikimedia.org 5
The nitrogen project Images courtesy of cfb.unh.edu, landcareresearch.com, geneticliteracyproject.org, edenbrothers.com 6
Engineering nitrogen-fixing E. coli ...faster characterization of the Cyanothece nitrogenase, which would be used to... ...engineer a Expressing a better minimal diazotrophic Cyanothece Synechosystis nif cluster in E. coli would lead to... Images courtesy of columbiariverkeeper.org, wikipedia, and ucdavis.edu 7
Nitrogen fixation is not easy for E. coli ● The nitrogenase reaction: 16 H 2 O + 16 ATP + 1 N 2 + 8 Reduced Flavodoxin → 16 ADP + 16 Phosphate + 2 NH 4 + 4 H + + 1 H 2 ● Cyanothece sp. ATCC 51142’s nif cluster is 35 contiguous genes ● Cyanothece promoters, transcription factors, and RBSs may be incompatible with E. coli 8
Project overview 9
Refining the nif cluster 2014 iGEM Team 2015 iGEM Team Native nif cluster - 35 genes Our designed nif cluster - 14 genes ● Regulatory Barriers ● Inducible Expression ● Unnecessary genes ● Optimized synthetic RBS ● Uncharacterized genes ● A Minimal Set of Genes 10
The minimal nif cluster ● 2 plasmids for simpler cloning ● Operons organized by expression level Structural Inducible promoter (pTrc) Inducible promoter (araBAD) Iron Molybdenum Synthesis Iron Sulfur Synthesis Strong RBS Weak RBS Nitrogenase Stabilization 11
CRISPR/dCas9 allows for targeted knockdowns ● Blocks gene transcription ● Knockdown genes on minimal cluster ● 3 sgRNAs per gene/promoter Qi, et al. Cell , 2013. 12
Overexpression plasmids ● 14 plasmids of nif genes ● Inducible promoter 13
Results ● Sequence-confirmed ● Sequence-confirmed ● Restriction-digest- minimal nif cluster plasmids CRISPR/dCas9 confirmed ○ araBAD-cysE2USVWZhesB- plasmids overexpression Kan-Rep101 ○ H1 ○ S1 plasmids ○ H3 ○ S2 ○ cce_0551 ○ D1 ○ W1 ○ cce_0552 ○ D2 ○ W2 ○ cce_0555 ○ K1 ○ Z1 ○ cce_0556 ○ K2 ○ Z2 ○ cce_0562 ○ K3 ○ hesB1 ○ cce_0566 ○ ara1 ○ hesB2 ○ cce_0567 ○ ara3 ○ hesB3 ○ hesA ○ E1 ○ Trc1 ○ nifB ○ E2 ○ Trc2 ○ nifEN ○ E3 ○ B2 ○ nifV ○ N2 ○ V2 ○ nifX ○ N3 14
Status of wet lab work Complete minimal Testing nif plasmids ● Acetylene reduction assay protocol ● First complete developed by 2014 ● Second on its way member Caroline Focht CRISPR/dCas9 Overexpression targeted knockouts plasmids ● 27 complete ● 12 complete ● 21 on their way ● 2 on their way 15
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
Main objectives Goal: Optimize nitrogen fixing E. coli using computational modeling • Task 1: Identify media supplements to increase ATP production and growth for N 2 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
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
In silico gene knockouts •Iteratively performing FBA •Double gene knockouts: computationally intensive •Want to couple metabolite production to biomass •No coupling found Single Gene Double Gene between flavodoxin Knockouts Knockouts reduction and biomass (50% of total) No effect on 1072 573070 biomass Some effect on 39 42383 biomass Lethal 255 40 19
Changes in pyruvate metabolism • Flux variability analysis : cells allocate more flux through pyruvate synthase (POR5) under N 2 fixing conditions • POR5 produces reduced flavodoxin • In silico pyruvate dehydrogenase knockout (PDH) = increased flux through POR5 • PDH KO leads to pyruvate buildup in cell 2 • Recommended in vivo PDH knockout, combined with POR5 overexpression (adapted from Voet, Voet) 20 2. U.S. National Library of Medicine. Result Filters. National Center for Biotechnology Information .
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
Part characterization collaboration with Vanderbilt iGEM ● Ran induction experiments to determine validity of the part ● Part K314100 + RFPyy did not work in either strain 22
Registry characterization ● Part is TetR-pTet system that expresses RFP when induced ● First transformation produced cells that were red in color K577895 in DH10B 23
BBa_K577895 characterization ● Performed a second transformation 24
BBa_K577895 characterization ● Performed a third induction experiment ● Results highlight the discrepancy in the two transformations 25
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
Special thanks to our sponsors... The Focht, Bourg, and Heeney/Toomey Families NSF-MCB Award #1331194 27
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
Learn more about our team and project 2015.igem.org/Team:WashU_StLouis washu.igem@gmail.com @WashUiGEM WashU iGEM Visit our poster: Hall C, No. 70 29
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