1 The world needs fixed nitrogen Average fertilizer input (kg ha - - PowerPoint PPT Presentation

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The world needs fixed nitrogen

World population (millions) % World population Average 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?

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Diagram courtesy of Wikimedia.org

The nitrogen project

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Images 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

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Images 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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Project

• verview

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

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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

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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

• verexpression

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 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

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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.

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Human practices

• Agriculture-focused panel presentation
• pen 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 Families NSF-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

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Visit our poster: Hall C, No. 70