Infant and Maternal Care Hunger and Nutrition Vitamin A Deficiency - - PowerPoint PPT Presentation

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Nov 24, 2022 28 likes •431 views

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

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Hunger and Nutrition Infant and Maternal Care Sustainability

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Vitamin A Deficiency in Pregnant Women World-wide

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Synthetic vitamin constructs Modeling and Optimization T

olkit

Quantification and Measurement Deployment

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Image of earl y Sumerian brewing techniques

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Pathways

Vitamin A Vitamin C

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Farnesyl Diphosphate

Geranyl-Geranyl Diphosphate

Phytoene

Neurosporene

Lycopene Beta- Carotene crtE crtYB crtI BTS1 β-Carotene (pro-vitamin A) BBa_K530000 BBa_K530001 BBa_K530002

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Wild-T ype Y east VitaY east

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β-carotene autofluoresces green

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Dough Media YPD

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GDP-D-Man GDP-L-Gal L-Gal-1- Phosphate α-L-Gal-1- Phosphate L-Gal-1,4- Lac L-Ascorbate

GME GME VTC2 VTC4 ARA1 ARA2 ALO1

GDP

L-ascorbate (vitamin C) BBa_K530025 BBa_K530026 BBa_K530027

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QuantiY east

Parts Characterization

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5E-10 1E-09 1.5E-09 2E-09 2.5E-09 3E-09 3.5E-09 20 40 60 80 100 120 140 160

β-carotene per cell (μg) Time (hours)

β-Carotene Purification and Spectrometry

WT crtE/YB/2I/tHMG1 crtE/YB/I/tHMG1 crtE/YB/2I crtE/YB/2I/tHMG1

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𝑤 = 𝑙𝑑𝑏𝑢 𝐹 𝑢 𝑇 𝑢 𝑙𝑁 + 𝑇 𝑢

Concentration Accumulation rate Enzyme assays 𝑙𝑑𝑏𝑢 𝑙𝑁 Reaction parameters

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Simulation Optimization

OptiY east

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Metabolic Pathway Expression

Enzymes Substrates Vitamins DNA

𝑒 𝑒𝑢 𝐹 = 𝛾 − 𝛽 𝐹 𝑒 𝑒𝑢 𝑇𝑗 = 𝑙𝑑𝑏𝑢

𝑘→𝑗 𝐹 𝑘 𝑇𝑗−1

𝑙𝑁

𝑘→𝑗 + 𝑇𝑗−1 𝑘

− 𝑙𝑑𝑏𝑢

𝑗→𝑙 𝐹𝑙 𝑇𝑗

𝑙𝑁

𝑗→𝑙 + 𝑇𝑗 𝑙

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0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 BTS1 crtE crtYB (GGPP) crtI (step 1) crtI (step 2) crtYB (neurosporene) crtYB (lycopene)

Relative Sensitivities Reaction Kcat Values

Sensitivities in Beta-Carotene Pathway

0.00 0.20 0.40 0.60 0.80 1.00 GDP clearance GME GME inhibition VTC2 VTC4 Relative Sensitivities

Model Parameters and Reaction Kcat Values

Sensitivities in L-Ascorbate Pathway

BTS1 and crtE are redundant, but crtE is more important Inhibition of GME by GDP does not strongly affect the model

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0.00E+00 1.00E-06 2.00E-06 3.00E-06 4.00E-06 5.00E-06 6.00E-06 7.00E-06 8.00E-06 9.00E-06 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

Beta-Carotene Produced (μM) Nitrogen Used (molecules)

Beta-Carotene Pareto Frontier

0.00E+00 1.00E-06 2.00E-06 3.00E-06 4.00E-06 5.00E-06 6.00E-06 7.00E-06 8.00E-06 9.00E-06 1.00E-05 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

L-Ascorbate Produced (μM) Nitrogen Used (molecules)

L-Ascorbate Pareto Frontier

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0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

BTS1 crtE crtYB crtI

Fraction of marginal nitrogen atom allocated Genes coding for metabolic enzymes

Fractional Nitrogen Allocation in an Optimal Beta-Carotene Pathway

0.1 0.2 0.3 0.4 0.5

GME VTC2 VTC4

Fraction of marginal nitrogen atom allocated Genes coding for metabolic enzymes

Fractional Nitrogen Allocation in an Optimal L-Ascorbate Pathway

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Y east T

olkit

Promoters UTRs Vectors Reporters

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8 Promoters 5 UTRs

BBa_K530004 BBa_K530005 BBa_K530006 BBa_K530007 BBa_K530008 BBa_K530009 BBa_K530010 BBa_K530012 BBa_K530014 BBa_K530015 BBa_K530016 BBa_K530017 BBa_K530019 BBa_K530020

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Integrating CEN/ARS 2-micron T ypes| Markers| HIS3 TRP1 LEU2 URA3

12 new vectors for yeast expression

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Reporters

L ycopene Beta- Carotene Violacein

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VitaBread

Baking with VitaY east

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8000 OD of VitaYeast (0.28 oz)

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HPLC results 450nm 𝑢𝑆𝑓𝑢 = 17.7𝑛𝑗𝑜

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HumanPractices

The real world

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Institutional Review Board (IRB)-Approved Survey

N = 30

5 10 15 20

Favorable Neutral Unfavorable

Number of People

General Attitude Toward GM Food Baltimore’s Northeast Market

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0-1% 1-21% 21-41% 41-61% 61-81% 81-100% Percent of People % Modified

Percent Modified Tolerance

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Accomplishments

BioBricked and

sequenced β-carotene and l-ascorbate pathways

BioBricked 8 promoters,

5 UTRs, and 12 yeast shuttle vectors

Utilized the LBS

modeling language for the first time in iGEM

Optimized β-carotene

and l-ascorbate production in silico

Distributed an IRB-

approved survey to local food markets

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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

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JHU Department of Chemical and Biomolecular Engineering JHU Department of Computer Science JHU Department of Biology JHU Student Initiatives Fund JHU School of Arts & Sciences JHU School of Engineering JHU Department of Biomedical Engineering JHU Department of Applied Mathematics & Statistics 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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