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The world needs fixed nitrogen

Wor

ld p

opul

atio

n (m

illio

ns)

% W

orld populationA

verage fertilizer input (kg ha⁻¹ yr⁻¹)

Year

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Erisman, JW, et al. Nature, 2008.

Problems with Haber Bosch● Energy-intensive● Creates

greenhouse gases● Fertilizer run-off

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An alternative to Haber Bosch

● Cyanothece sp. 51142 photosynthesizes and fixes nitrogen○ Separates them temporally

● Nitrogenase enzyme

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need reference

Bandyopadhyay, et al. Nature, 2010.

From Cyanothece to plants?

● Cyanothece related to chloroplasts

● Can we engineer nitrogen-fixing plants?

5Diagram courtesy of Wikimedia.org

The nitrogen project

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

Expressing a minimal Cyanothece nif cluster in E. coli would lead to...

...faster characterization of the Cyanothece nitrogenase, which would be used to...

...engineer a better diazotrophic Synechosystis

Engineering nitrogen-fixing E. coli

7Images courtesy of columbiariverkeeper.org, wikipedia, and ucdavis.edu

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 51142’s nif cluster is 35 contiguous genes

● Cyanothece promoters, transcription factors, and RBSs may be incompatible with E. coli

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Projectoverview

Refining the nif cluster

2014 iGEM TeamNative nif cluster - 35 genes ● Regulatory Barriers● Unnecessary genes● Uncharacterized genes

2015 iGEM TeamOur designed nif cluster - 14 genes ● Inducible Expression● Optimized synthetic RBS● A Minimal Set of Genes

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The minimal nif cluster

● 2 plasmids for simpler cloning● Operons organized by expression level

Structural Iron Molybdenum Synthesis Iron Sulfur Synthesis Nitrogenase Stabilization 11

Inducible promoter (pTrc)

Strong RBS

Inducible promoter (araBAD)

Weak RBS

CRISPR/dCas9 allows for targeted knockdowns● Blocks gene transcription● Knockdown genes on

minimal cluster● 3 sgRNAs per

gene/promoter

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Qi, et al. Cell, 2013.

Overexpression plasmids

● 14 plasmids of nif genes

● Inducible promoter

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Results

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● Sequence-confirmed CRISPR/dCas9 plasmids

○ H1○ H3○ D1○ D2○ K1○ K2○ K3○ ara1○ ara3○ E1○ E2○ E3○ N2○ N3

● Sequence-confirmed minimal nif cluster plasmids

○ araBAD-cysE2USVWZhesB-Kan-Rep101 ○ S1

○ S2○ W1○ W2○ Z1○ Z2○ hesB1○ hesB2○ hesB3○ Trc1○ Trc2○ B2○ V2

● Restriction-digest-confirmed overexpression plasmids

○ cce_0551○ cce_0552○ cce_0555○ cce_0556○ cce_0562○ cce_0566○ cce_0567○ hesA○ nifB○ nifEN○ nifV○ nifX

Status of wet lab work

CRISPR/dCas9 targeted knockouts

● 27 complete● 21 on their way

Complete minimal nif plasmids

● First complete● Second on its way

Testing● Acetylene reduction

assay protocol developed by 2014 member Caroline Focht

Overexpression plasmids

● 12 complete● 2 on their way

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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)

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Main objectivesGoal: 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

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Media supplementation ● Supplemented glucose in

existing media with additional substrate equivalent to 60

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● Identified 15 metabolites as having:

● Largest increase in ATP production per increase in max biomass

● Larger increases in ATP production than additional glucose

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

1072 573070

Some effect on biomass

39 42383

Lethal 255 40

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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

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

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Part characterization collaboration with Vanderbilt iGEM

● Ran induction experiments to determine validity of the part

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● Part K314100 + RFPyy did not work in either strain

Registry characterization

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K577895 in DH10B

● Part is TetR-pTet system that expresses RFP when induced

● First transformation produced cells that were red in color

BBa_K577895 characterization

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● Performed a second transformation

BBa_K577895 characterization

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● Performed a third induction experiment

● Results highlight the discrepancy in the two transformations

Added 12 new composite RBS parts to the registry

● RBSs used for genes in minimized nif cluster

● Differing levels of expression

● On each BioBrick:○ Constitutive pTet

promoter○ RBS○ mRFP○ Two terminators

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Special thanks to our sponsors...

The Focht, Bourg, and Heeney/Toomey FamiliesNSF-MCB Award #1331194

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Special thanks to our mentors...

(Left to right) Carlos Barba, Cheryl Immethun, Yi Xiao, Andrea Balassy, Thomas Mueller, Young Je Lee, Ray Henson, Caroline Focht

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Tae Sook Moon Fuzhong Zhang Costas Maranas

Learn more about our team and project

2015.igem.org/Team:WashU_StLouis

washu.igem@gmail.com

@WashUiGEM

WashU iGEM

29Visit our poster: Hall C, No. 70