Washington University’s first iGEM team Food and Energy Track
Introduc=on
Life in a Photobioreactor Large Light Harves=ng Antenna Small Light Harves=ng Antenna
Life in a Photobioreactor Theore=cal Energy Wasted Energy Through NPQ Produc=on Photosynthe=c Satura=on Curve Energy Produced
ENERGY WASTED PRODUCED
ENERGY WASTED PRODUCED
Leaf Size in the Eastern Black Oak etc High Branch Middle Branch Lower Branch David Sibley‐ “The Sibley Guide to Trees
ENERGY PRODUCED ENERGY WASTED
ENERGY PRODUCED ENERGY WASTED
The Project
The Organism New chassis for synthe=c biology • Rhodobacter sphaeroides is a purple • Alphaproteobacteria. Metabolically flexible: • – aerobic and anaerobic respira=on – Phototrophic under anaerobic condi=ons with light. R. sphaeroides is one of the best understood • photosynthe=c organisms. – photosystem is located in intracytoplasmic membrane invagina=ons – Light Hares=ng Complex 2 (LH2) – Light Harves=ng Complex 1 (LH1) – Reac=on Center (RC). – These pigment‐protein complexes non‐ covalently bind bacteriochlorophylls and carotenoids.
Light Harves=ng Antenna 2 • LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm • Funnels its energy to LH1 and the reac=on center for photochemistry. • The two subunits of LH2 are coded for by the pucB/A genes • Naturally promoted by the puc promoter.
Wild Type High Oxygen Low Oxygen • Puc promoter downregulated • Transcrip=on from the puc promoter is high • No expression of pucB/A and thus LH2 • pucB/A expressed, high LH2 expression (big antenna complex)
Synthe=c Regula=on of pucB/A Puc Promoter cph8 ompR RBS RBS ompC Promoter pubB/A RBS pucC pRKCBC3
Mutant • Under Low light intensi=es Cph8 ac=ve and • Keep under low oxygen tension OmpR phosphorylated, leading to puc B/A and • Cph8 light sensor under control of puc LHII expression promoter, and puc B/A genes behind OmpC • LH2 Expression is inversely correlated to light promoter Intensity • High light intensi=es repress OmpR phosphoryla=on and puc B/A expression
Parts • Submibed 10 parts to the registry • Plan to submit 2 more in the near future • 10 total R. sphaeroides specific parts • Constructed 4 other parts that aren’t compa=ble with Registry Standards
Parts pucB/A as a reporter • LH2 absorp=on at 800 and 850nm is absent in LH2 deficient mutant DBC Ω • Indicates its efficacy as a reporter. • Expression is higher from genomic DNA than on pRKCBC3 • Indicates that can use pRKCBC3 + pucPromoter and pucB/A as truncated antenna
Parts Strength of the puc Promoter • The puc promoter is down‐regulated at high oxygen tension • Nearly cons=tuent at low oxygen tension
Tissue Flask Experiment Designed this experiment to examine: • – how available light influences growth on a series of bioreactors the effect of Non‐Photochemical Quenching and photodamage – The first group to measure these perameters • Light that passes through this flask is the only source of light for those flasks • behind it • Conducted experiment on R. sphaeroides 2.4.1 and R. sphaeroides DBCΩ (LH2 Knockout) Measured Growth Rates (OD 600) using a spectrophotometer • Measured the absolute irradiance of light incident on the flasks using a • spectroradiometer
Absolute Irradiance on Flask 2 Wild Type DBCΩ
Absolute Irradiance on Flask 3 Wild Type DBCΩ
Absolute Irradiance on Flask 4 Wild Type DBCΩ
Absolute Irradiance on Flask 5 Wild Type DBCΩ
Flask 1 Growth Wild Type DBCΩ 2.5 2.5 2 2 1.5 1.5 1 1 0.5 0.5 0 0 Day 0 Day 0 Day 1 Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Day 4 Day 5 Day 5 Growth over 5 days at OD 600 for Flask 1 • WT absorbs LH2 .987 • As such, it can be wavelength light reasoned that .967 at 800 and 850 nm NPQ is occurring • Yet WT grows the as the photons same amount as absorbed by LH2 DBCΩ don’t appear to affect growth
Flask 2 Growth Wild Type DBCΩ 2.5 2.5 2 2 1.5 1.5 1 1 0.5 0.5 0 0 Day 0 Day 0 Day 1 Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Day 4 Day 5 Day 5 Growth over 5 days at OD 600 for Flask 2 • WT second flask • DBCΩ flask 2 >2.5 grew extremely grew less than well flask 1 • Appears that • Likely due to photodamage also abenuated light at occurred in WT LH1 870 nm .512 flask 1 as it grew wavelength from less then flask 2 first flask
Flask 3 Growth Wild Type DBCΩ 2.5 2.5 2 2 1.5 1.5 1 1 0.5 0.5 0 0 Day 0 Day 0 Day 1 Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Day 4 Day 5 Day 5 Growth over 5 days at OD 600 for Flask 3 • WT growth is at .272 • DBCΩ flask 3 the same rate as .271 grew less than DBCΩ flask 1 and 2 • Light available at • Also due to LH2 wavelengths abenuated light at is depleted LH1 870 nm • does not wavelength contribute to growth
Flask 4 Growth Wild Type DBCΩ 2.5 2.5 2 2 1.5 1.5 1 1 0.5 0.5 0 0 Day 0 Day 0 Day 1 Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Day 4 Day 5 Day 5 Growth over 5 days at OD 600 for Flask 4 Dark Growth • WT is at .209 (Heterotrophic) heterotrophic growth levels OD Day 5 • DBCΩ is s=ll .124 2.4.1 .122 growing photosynthe=cally DBCΩ .151
Flask 5 Growth Wild Type DBCΩ 2.5 2.5 2 2 1.5 1.5 1 1 0.5 0.5 0 0 Day 0 Day 0 Day 1 Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Day 4 Day 5 Day 5 Growth over 5 days at OD 600 for Flask 5 Dark Growth • WT is at (Heterotrophic) .208 heterotrophic growth levels OD Day 5 • DBCΩ is s=ll 2.4.1 .122 .110 growing photosynthe=cally DBCΩ .151
Cumula=ve Growth The CumulaFve Growth of DBCΩ The CumulaFve Growth of Wild Type Tissue Flasks Tissue Flasks • Cumula=ve growth of the =ssue flasks as measured by the sum of the op=cal densi=es of the respec=ve cell type's =ssue flasks at a given day • The contribu=on of a given =ssue flask to cumula=ve growth is displayed.
Conclusions of Tissue Flask Experiment In Our Mutant • Photodamage occurred in WT Flask 1 • NPQ quenching occurred in WT Flask 1 • LH2 Wavelength light in the WT was depleted aoer Flask 2 • All photosynthe=c growth in the WT flasks was absent aoer Flask 3 • All DBCΩ flasks grew photosynthe=cally and the amount of growth was inversely correlated to depth in the bioreactor • Cumula=ve WT flask growth is greater than DBCΩ The size of the Light Harves=ng Antenna (LH2) is inversely correlated to light intensity‐ As such, these effects should not be observed and growth should be greater than the wild type
Modeling our Mutant vs. the WT
• In a bioreactor, cells at the surface absorb more than enough light to saturate their photosynthe=c apparatus, transmipng less energy to deeper layers. Nonlinear LS EsFmaFon of WT Total SaturaFon Curve • For wild type cells, the satura=on curve is approximately the same for all cells, regardless of their incident light intensity.
• For our mutant cells, this curve scales as a func=on of light intensity, due to nega=ve regula=on of LH2 complex produc=on • For our mutant cells, this curve scales as a func=on of light intensity, due to nega=ve regula=on of LH2 complex produc=on
AssumpFons • Satura=on curve: Absorbance as a func=on of incident light intensity. The coefficient changes with intensity in the mutant only. • Light intensity at next layer is given by transmibance from previous layer (assume no backscabering). • Total energy funneled to photosynthe=c pathways es=mated sum of light absorbed by each layer.
Revisions based on empirical data • Background LH1 absorbance • Divide Mutant by correc=on factor (1 ‐ 0.2) Growth over 5 days at OD 600 for Flask 1 • PhotoinhibiFon • Limit first flask absorbance to 1
Layer 1
Layer 2
Layer 3
Layer 4
Layer 5 Absorbance Wild Type Mutant Mean 0.389 0.476 Std. Dev. 0.480 0.375
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