WashU iGEM 2014 Team nitro GENIUS ! Washington University in St. - - PowerPoint PPT Presentation

WashU iGEM 2014 Team nitroGENIUS!

Washington University in St. Louis

Meet Team nitroGENIUS!

Richard Li Benjamin Huang Jeffrey Lee Caroline Focht

Andrew Ng Bert Berla Deng Liu Cheryl Immenthun

Meet the Team nitroGENIUS! Advisors

• Dr. Pakrasi
• Dr. Moon

Meet the Team nitroGENIUS! Advisors

The World, Nitrogen, and the Future

The World We Live In

805 million people in the world do not have enough to eat Poor nutrition causes nearly half (45%) of deaths in children under five

Statistics provided by the United Nations World Food Programme Picture courtesy

• f solarviews.com

Nitrogen inequality in agriculture

Data taken from http://archive.bio.ed.ac.uk/jdeacon/microbes/nitrogen.htm

Team nitroGENIUS reaches out Raising Awareness

Like, Comment, Subscribe @ www.youtube.com/WashUiGEM

The Problem with Nitrogen Fixation

O2 reacts with the metal core and deactivates the enzyme

Picture from: Dixon, Kahn. Genetic Regulation of Biological Nitrogen Fixation. Nature Reviews. August 2004, Vol 2. P. 621-631.

The BIG Picture

Organism Ease of Engineering Photosynthetic Crop Plant

• E. coli

✓✓ ✕ ✕ Cyanobacteria ✓ ✓ ✕ Chloroplast ✕ ✓ ✓

• E. coli

Pictures taken from: www.pitch.com, http://protist.i.hosei.ac.jp, and http://www.motherearthnews.com

Photosynthetic Cyanothece sp. 51142 also fixes nitrogen through a circadian rhythm

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

Cyanothece sp. ATCC 51142

Figure 1. Unicellular Cyanobacteria Fixes its own Nitrogen

Nitrogenase is irreversibly inactivated by oxygen

NH3

Transfer Cyanothece nif cluster into E. coli Transfer Synechocystis light regulation into E. coli Light regulated nif cluster in E. coli

Project Overview

Getting the Cyanothece sp. 51142 nif cluster to function in E. coli.

Cyanothece sp. 51142 nif cluster

• Large size—34 genes carried in

38,000 bp plasmid

• Functionally complete nif cluster
• Burdensome in terms of energy

and material consumption

Figure above adapted from: Bandyopadhyay A., et al. Mbio. 2011. 2(5). Doi: 10.1128/mBio.00214-11

Different parameters were tested to find the

• ptimal conditions for experimentation
• E. coli Strains: Top 10, WM1788, DH5α,

JM109, BL21

• Conditions:

○ Growth Media: LB or M9 ○ Nitrogen Sources: Glutamine, Glutamate, Ammonium Chloride ○ O2 level: Aerobic vs. Anaerobic ○ Temperature: 30℃ vs 37℃

0" 5" 10" 15" 20" 25" 30" 35" 40" 45" 50" 55" 60" 65" 0" 10" 20" 30" 40" 50" 60" 70" 80"

C2H2""->"C2H4"(x"103")" Time"(hours)" Nitrogen"FixaAon"AcAvity"Vs."Time"in"E.#coli#WM1788"and"JM109"

WM1788"WT" JM109"WT"

• E. coli fixes nitrogen!

0" 5" 10" 15" 20" 25" 30" 35" 40" 45" 50" 55" 60" 65" 0" 10" 20" 30" 40" 50" 60" 70" 80"

C2H2""->"C2H4"(x"103")" Time"(hours)" Nitrogen"FixaAon"AcAvity"Vs."Time"in"E.#coli#WM1788"and"JM109"

WM1788"w/"Plasmid"" JM109"w/"Plasmid" WM1788"WT" JM109"WT"

Future Directions

• Determine a minimal nif cluster
• Test minimized nif cluster
• “pRF minimal cluster”
• Further optimize conditions
• Test more options for known parameters
• Identify and optimize newly identified parameters

Creating a light repression mechanism to turn off transcription of nif in the light

CcaR

Synechocystis sp. PCC6803’s CcaR/CcaS light-activated response regulator

Light source Chromophore CcaS CcaR Activated by light Autophosphorylation G- box Pcpcg2 +1

• 108
• 10
• 35

Information adapted from: Hirose, et al. PNAS., 2008 July 15; vol. 105 No. 28

PcpcG2’s dynamic range in E. coli between on and off states is poor

Figures both adapted from: Tabor, et al. J. Mol. Biol. 2011

Dark Light Fluorescence (Miller Units)

Ptrc1O is a strong promoter in both E. coli and Synechocystis

Adapted from: Huang, et al. Nucleic Acids Research. 2010

BBa_K1385001

Our hybrid promoter aims to combine the light regulation of PcpcG2 with the strength of Ptrc1O

Constructs

BBa_K1385000 Pcpcg2 +1

• 108

G- box

• 10
• 35

Binding region of CcaR PcpcG2 TetR Phybrid +1

• 108

G- box

• 10
• 35

Binding region of CcaR Ptrc1O Phybrid TetR

TetR binds reversibly to PLTetO1, which should turn off EYFP expression in the light

Experimental Constructs Dark Light Chromophore Bba_K1017726 N/A N/A Positive Control ON ON Negative Control OFF OFF PBJ003 ON OFF HYB001 ON OFF

PLtetO-1 EYFP PcpcG2 Nothing PBBa_J23101 PcyA ho1 PcpcG2 TetR PLtetO-1 EYFP PcpcG2 TetR PLtetO-1 EYFP

Light induction experiment. Preliminary Results: both promoters are leaky

Adding anhydrotetracycline (aTc): Hybrid system seems to have greater dynamic range

~30x Fold Change ~3x Fold Change

Future Directions:

• Promoters are both leakier than expected
• Weaken RBS of TetR
• Find tightest on-off without having to induce with aTc
• Swap EYFP for nif cluster

PcpcG2 TetR PLtetO-1 nif

Baby Steps

Organism Ease of Engineering Photosynthetic Crop Plant

• E. coli

✓✓ ✕ ✕ Cyanobacteria ✓ ✓ ✕ Chloroplast ✕ ✓ ✓

• E. coli

Pictures taken from: www.pitch.com, http://protist.i.hosei.ac.jp, and http://www.motherearthnews.com

Special thanks to our funding sources, National Science Foundation MCB and the following sponsors:

washu.igem@gmail.com http://www.facebook.com/washuigem #teamnitroGENIUS

Questions?

Questions?