Sustainability Infant and Maternal Care Hunger and Nutrition
Vitamin A Deficiency in Pregnant Women World-wide
Quantification and Measurement Synthetic T oolkit Deployment Modeling and vitamin Optimization constructs
Image of earl y Sumerian brewing techniques
Pathways Vitamin A Vitamin C
β -Carotene (pro-vitamin A) Farnesyl Diphosphate crtE BTS1 Geranyl-Geranyl Diphosphate BBa_K530000 Phytoene BBa_K530001 crtYB crtI Neurosporene BBa_K530002 Lycopene Beta- Carotene
Wild-T ype Y east VitaY east
β -carotene autofluoresces green
Dough Media YPD
L-ascorbate (vitamin C) GDP-D-Man GME GME GDP-L-Gal BBa_K530025 VTC2 GDP BBa_K530026 L-Gal-1- Phosphate BBa_K530027 VTC4 α -L-Gal-1- Phosphate ARA1 ARA2 L-Gal-1,4- Lac ALO1 L-Ascorbate
QuantiY east Parts Characterization
β -Carotene Purification and Spectrometry 3.5E-09 3E-09 β -carotene per cell ( μ g) 2.5E-09 2E-09 1.5E-09 1E-09 5E-10 0 0 20 40 60 80 100 120 140 160 Time (hours) WT crtE/YB/2I/tHMG1 crtE/YB/I/tHMG1 crtE/YB/2I crtE/YB/2I/tHMG1
Enzyme assays Reaction parameters Concentration 𝑤 = 𝑙 𝑑𝑏𝑢 𝐹 𝑢 𝑇 𝑢 Accumulation rate 𝑙 𝑁 + 𝑇 𝑢 𝑙 𝑁 𝑙 𝑑𝑏𝑢
OptiY east Simulation Optimization
Expression DNA 𝑒 𝑒𝑢 𝐹 = 𝛾 − 𝛽 𝐹 Metabolic Enzymes Pathway Substrates Vitamins 𝑘→𝑗 𝐹 𝑗→𝑙 𝐹 𝑙 𝑇 𝑗 𝑒𝑢 𝑇 𝑗 = 𝑙 𝑑𝑏𝑢 𝑒 𝑘 𝑇 𝑗−1 − 𝑙 𝑑𝑏𝑢 𝑘→𝑗 + 𝑇 𝑗−1 𝑗→𝑙 + 𝑇 𝑗 𝑙 𝑁 𝑙 𝑁 𝑘 𝑙
Sensitivities in Beta-Carotene Pathway 1.00 0.90 Relative Sensitivities 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 BTS1 crtE crtYB (GGPP) crtI (step 1) crtI (step 2) crtYB (neurosporene) crtYB (lycopene) Reaction Kcat Values BTS1 and crtE are redundant, but crtE is more important Sensitivities in L-Ascorbate Pathway 1.00 Relative Sensitivities 0.80 0.60 0.40 0.20 0.00 GDP clearance GME GME inhibition VTC2 VTC4 Model Parameters and Reaction Kcat Values Inhibition of GME by GDP does not strongly affect the model
Beta-Carotene Pareto Frontier 9.00E-06 Beta-Carotene Produced ( μ M) 8.00E-06 7.00E-06 6.00E-06 5.00E-06 4.00E-06 3.00E-06 2.00E-06 1.00E-06 0.00E+00 0.E+00 5.E+04 1.E+05 2.E+05 2.E+05 3.E+05 3.E+05 4.E+05 4.E+05 Nitrogen Used (molecules) L-Ascorbate Pareto Frontier 1.00E-05 9.00E-06 L-Ascorbate Produced ( μ M) 8.00E-06 7.00E-06 6.00E-06 5.00E-06 4.00E-06 3.00E-06 2.00E-06 1.00E-06 0.00E+00 0.E+00 1.E+05 2.E+05 3.E+05 4.E+05 5.E+05 6.E+05 7.E+05 8.E+05 9.E+05 Nitrogen Used (molecules)
Fractional Nitrogen Allocation in an Optimal Beta-Carotene Pathway 0.5 0.45 nitrogen atom allocated Fraction of marginal 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 BTS1 crtE crtYB crtI Genes coding for metabolic enzymes Fractional Nitrogen Allocation in an Optimal L-Ascorbate Pathway 0.5 nitrogen atom allocated 0.4 Fraction of marginal 0.3 0.2 0.1 0 GME VTC2 VTC4 Genes coding for metabolic enzymes
Y east T oolkit Promoters UTRs Vectors Reporters
BBa_K530004 BBa_K530005 BBa_K530006 BBa_K530007 BBa_K530008 8 Promoters 5 UTRs BBa_K530009 BBa_K530010 BBa_K530012 BBa_K530014 BBa_K530015 BBa_K530016 BBa_K530017 BBa_K530019 BBa_K530020
12 new vectors for yeast expression T ypes| Integrating CEN/ARS 2-micron Markers| HIS3 TRP1 LEU2 URA3
Reporters Beta- L ycopene Violacein Carotene
VitaBread Baking with VitaY east
8000 OD of VitaYeast (0.28 oz)
HPLC results 450nm 𝑢 𝑆𝑓𝑢 = 17.7𝑛𝑗𝑜
HumanPractices The real world
Institutional Review Board (IRB)-Approved Survey General Attitude Toward GM Food 20 Number of People 15 N = 30 10 5 0 Favorable Neutral Unfavorable Percent Modified Tolerance 100% 90% 80% Percent of People 70% 60% Baltimore’s Northeast Market 50% 40% 30% 20% 10% 0% 0-1% 1-21% 21-41% 41-61% 61-81% 81-100% % Modified
Accomplishments • BioBricked and • Utilized the LBS sequenced modeling language for β -carotene and the first time in iGEM l-ascorbate pathways • Optimized β -carotene • BioBricked 8 promoters, and l-ascorbate 5 UTRs, and 12 yeast production in silico shuttle vectors • Distributed an IRB- approved survey to local food markets
More to Come • Attempt to achieve predicted optimum enzyme levels using our expression cassette library • Search for additional funding to continue research and deploy our work in the field
JHU Department of JHU Department of Biomedical Engineering Chemical and Biomolecular Engineering JHU Department of JHU Department of Computer Science Applied Mathematics & Statistics JHU Department of JHU Student JHU School of JHU School of Biology Initiatives Fund Engineering Arts & Sciences Special thanks to our advisors and mentors Dr. Jef Boeke | Dr. Marc Ostermeier | Dr. Joel Bader | Dr. Mike Betenbaugh | Dr. T akanari Inoue Dr. Deb Mathews | Dr. Patrick Cai | Dr. Leslie Mitchell | Dr. Giovanni Stracquadanio | Marty T aylor
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