WashU iGEM 2014 Team nitro GENIUS ! Washington University in St. Louis
Meet Team nitro GENIUS! Benjamin Huang Jeffrey Lee Caroline Focht Richard Li
Meet the Team nitro GENIUS! Advisors Cheryl Immenthun Andrew Ng Bert Berla Deng Liu
Meet the Team nitro GENIUS! Advisors Dr. Moon Dr. Pakrasi
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 Picture courtesy Statistics provided by the United of solarviews.com Nations World Food Programme
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 O 2 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 E. coli Organism Ease of Engineering Photosynthetic Crop Plant ✓✓ E. coli ✕ ✕ ✓ ✓ Cyanobacteria ✕ ✓ ✓ Chloroplast ✕ 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 Cyanothece sp. ATCC 51142 N 2 + 8H + + 8e - + 16ATP 2NH 3 + H 2 + 16ADP + 16Pi NH 3 Figure 1. Unicellular Cyanobacteria Fixes its own Nitrogen Nitrogenase is irreversibly inactivated by oxygen
Project Overview Transfer Cyanothece nif cluster into E. coli Light regulated nif cluster in E. coli Transfer Synechocystis light regulation into E. coli
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 optimal conditions for experimentation ● E. coli Strains : Top 10, WM1788, DH5 α , JM109, BL21 ● Conditions : ○ Growth Media: LB or M9 ○ Nitrogen Sources: Glutamine, Glutamate, Ammonium Chloride ○ O 2 level: Aerobic vs. Anaerobic ○ Temperature: 30 ℃ vs 37 ℃
E. coli fixes nitrogen! Nitrogen"FixaAon"AcAvity"Vs."Time"in" E.#coli# WM1788"and"JM109" Nitrogen"FixaAon"AcAvity"Vs."Time"in" E.#coli# WM1788"and"JM109" 65" 65" 60" 60" WM1788"w/"Plasmid"" 55" 55" JM109"w/"Plasmid" 50" 50" C 2 H 2 ""->"C 2 H 4 "(x"10 3 ")" C 2 H 2 ""->"C 2 H 4 "(x"10 3 ")" WM1788"WT" WM1788"WT" 45" 45" JM109"WT" JM109"WT" 40" 40" 35" 35" 30" 30" 25" 25" 20" 20" 15" 15" 10" 10" 5" 5" 0" 0" 0" 0" 10" 10" 20" 20" 30" 30" 40" 40" 50" 50" 60" 60" 70" 70" 80" 80" Time"(hours)" Time"(hours)"
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
Synechocystis sp. PCC6803’s CcaR/CcaS light-activated response regulator Activated by light Light source Chromophore Autophosphorylation CcaR CcaS CcaR P cpcg2 G- box -108 -35 -10 +1 Information adapted from: Hirose, et al. PNAS., 2008 July 15; vol. 105 No. 28
P cpcG2 ’s dynamic range in E. coli between on and off states is poor Fluorescence (Miller Units) Dark Light Figures both adapted from: Tabor, et al. J. Mol. Biol. 2011
P trc1O is a strong promoter in both E . coli and Synechocystis Adapted from: Huang, et al. Nucleic Acids Research. 2010
Our hybrid promoter aims to combine the light regulation of P cpcG2 with the strength of P trc1O Constructs +1 - 108 -10 - 35 P cpcg2 G- box Binding region of CcaR BBa_K1385000 P cpcG2 TetR +1 - 108 - 35 -10 P hybrid P trc1O G- box Binding region of CcaR BBa_K1385001 P hybrid TetR
TetR binds reversibly to P LTetO1 , which should turn off EYFP expression in the light Experimental Constructs Dark Light Chromophore N/A N/A Bba_K1017726 P BBa_J23101 PcyA ho1 Positive ON ON Control P LtetO-1 EYFP Negative OFF OFF Control P cpcG2 Nothing PBJ003 ON OFF P cpcG2 TetR P LtetO-1 EYFP HYB001 ON OFF P cpcG2 TetR P LtetO-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 P cpcG2 TetR P LtetO-1 nif
Baby Steps E. coli Organism Ease of Engineering Photosynthetic Crop Plant ✓✓ E. coli ✕ ✕ ✓ ✓ Cyanobacteria ✕ ✓ ✓ Chloroplast ✕ Pictures taken from: www.pitch.com, http://protist.i.hosei.ac.jp, and http://www.motherearthnews.com
Questions? Special thanks to our funding sources, National Science Foundation MCB and the following sponsors: Questions? washu.igem@gmail.com http://www.facebook.com/washuigem #teamnitroGENIUS
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