5-18-15 mona shores talk
TRANSCRIPT
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Undergraduate Research Summary and Current State-of-the-Art in Biotechnology
May 18, 2015
David Boyer
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Overview - Outline
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1. Introduction – Short Bio
2. Undergraduate Research Summary
a) Biofuels – E. coli Isobutanol Tolerance
b) Biofuels – Algae Hydrothermal Liquefaction Recycling
c) Enzymology/Green Chemistry – Ferulic Acid Decarboxylase
d) Synthetic Biology Team – Antibody Secretion in E. coli
3. Current Trends in Biotechnology
a) DNA Sequencing/Synthesis
b) DNA Editing Technology – CRISPR/Cas9
c) Protein Evolution/Engineering
d) Virus Encoded Batteries
e) Synthetic Microbial Consortium
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Introduction
Who am I? (Good Question)
Mona Shores High School – 2010University of Michigan BS (Honors) Biochemistry and Chemistry – 2014Ecovia Renewables LLC – Jan. 2015 – Present University of California Los Angeles – PhD Biochemistry (~2020 expected)General Interests: Music, Piano, Tennis, Running, Livin’ the Dream, etc.Research Interests: Biotechnology, Synthetic Biology, Chemistry, BiologyEmail: [email protected]
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Biofuels – General Overview
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Cellulosic Biomass (e.g., switchgrass)
Conversion of glucose to fuel through metabolic engineering
Large-scale production and isolation of fuels
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Biofuels Overview – Isobutanol Tolerance
5 out of 34Atsumi, et al. (2008) Nature.
James Liao: Isobutanol producing strain of E. coli derived from sugars and amino acid biosynthesis pathway
Problem: However, isobutanol is toxic to microbes that grow it!
Solution: Evolution and Genetic Engineering!
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Isobutanol Tolerance Project
6 out of 34Minty et al. Microb. Cell. Fact. 2011 10:18
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Genome Evolution: ResultsMechanisms of adaptation
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Regulatory - stress response attenuation:
Minty et al. Microb. Cell. Fact. 2011 10:18
Evolution: MIC increased from 1% to 2% w/v, 144x mutations across 6x lineages
Epistatic interactions: Positive epistasis between hfq and subsequent mutations
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Genome EngineeringOverview
8 out of 34(George Church and Harris Wang)Minty et al. Microb. Cell. Fact. 2011 10:18
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Algae Biofuels - Overview
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Algae Hydrothermal Liquefaction:Recycling Aqueous Waste
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Algae Hydrothermal Liquefaction:Recycling Aqueous Waste
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Algae Regrowth on AqAl
AqAl
AqAl SBM
AqAl SBM in Stripped COMBO
Bacterial Strains Grown/Removed from AqAl SBM
Processed AqAl SBM in Stripped
COMBO
Generate AqAl from 20% Algae Slurry
Make simple bacterial media (pH7 AqAl at ~20% +micronutrients)
Grow both parent and evolved strains in SBM
Dilute SBM into COMBO as sole
N/P source
Compare Algae growth rate and
max yield
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“processed” “raw”
• Incubated cultures of 30 vol% AqAl SBM with E. coli strains (“processed”), used “Steriflip” to filter out cells
• Same treatment with a “blank” (“raw”)
“Steriflip” Filtration Add Algae Inoculum
-Run Growth Tests with algae instripped COMBO (no C, N, P) at variousconcentrations of “raw” and “processed” AqAl SBM
Algae Hydrothermal Liquefaction:Recycling Aqueous Waste
Grow bacteria on Biofuels waste, feed it back to algae!
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Co-enzymes: Background
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ATP – Energy Source
FAD – Electron Carrier
Vitamin B12 – Radical Chemistry
Coenzyme A – Localization, trans-acetylation
Handle Moiety – For RecognitionBusiness End – Chemically Reactive
Enzymes are great, but they often need help!
Limited to chemical reactivity of ~20 amino acids, additional “co-enzymes” are used for certain chemistries
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Ferulic Acid Decarboxylase (FDC)
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Ferulic Acid Decarboxylase from S. cerevisiae (yeast) is one such enzyme that needs a cofactor (recently discovered!)
Another enzyme called Phenylacrylic Acid Decarboxylase shown to make this co-factor
Lin, F., Ferguson, K., Boyer, D., Lin, X. & Marsh, E. ACS Chem. Biol. (2015).
Activity of enzyme increases when unknown cofactor introduced
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Ferulic Acid Decarboxylase (FDC)Liquid Chromatography – Mass Spec
15 out of 34Boyer, David. Honors Thesis. deepblue.lib.umich.edu. (2014)
Protein Expression, Purification, Co-factor Isolation and Identification
LC-MS both separates compounds and determines their mass to charge ratio (i.e., their mass)
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Ferulic Acid Decarboxylase (FDC)Kinetic and Mechanistic Studies
16 out of 34Boyer, David. Honors Thesis. deepblue.lib.umich.edu. (2014)
Ultraviolet and Visible Light Spectrophotometry allows monitoring of reaction progress – can determine rates of reactions
Can use Nuclear Magnetic Resonace (H1-NMR) to determine which atoms are reactive – helps to determine mechanism
Use deuterium (H2) to determine which peaks disappear after reaction – those atoms are exchanged with solvent
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Ferulic Acid Decarboxylase (FDC)Kinetic and Mechanistic Studies
17 out of 34Boyer, David. Honors Thesis. deepblue.lib.umich.edu. (2014)
Hypothesized Mechanism
Computationally Predicted Mechanism
Reaction Energy Diagram
Can use computational chemistry software to help predict reaction mechanisms and transition state energies
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Ferulic Acid Decarboxylase (FDC)Kinetic and Mechanistic Studies
18 out of 34Boyer, David. Honors Thesis. deepblue.lib.umich.edu. (2014)
Recently Published Mechanism
Computationally Predicted Mechanism
Published mechanism (2015) basically identical to computationally predicted mechanism. Identifies key amino acids that are responsible catalysis.
Bhuiya et al. Applied and Environmental Microbiology (2015).
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Ferulic Acid Decarboxylase (FDC)Structural Studies: X-ray Crystallography
19 out of 34Bhuiya et al. Applied and Environmental Microbiology (2015).
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iGEM – International Genetically EngineeredMachine
20 out of 34www.igem.org
What is iGEM?
Teams from around the world work on summer synthetic biology projects (e.g., taking pictures with bacteria, detecting arsenic in drinking supplies) and meet in Boston to compete
Yours truly with Michigan Synthetic Biology team 2014 in Boston
Taking pictures with bacteria!
Out project – making antibody fragments in E. coli!
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iGEM – International Genetically EngineeredMachine
21 out of 34http://2014.igem.org/Team:Michigan
Eukaryotic cells have specific cellular compartments (e.g., Endoplasmic Reticulum) for folding of complex proteins (e.g., antibodies)
Excreting recombinantly expressed proteins into periplasmic space of E. coli allows folding of complex proteins
We used a “secretion tag” called OsmY. Synthesize a DNA sequence that contains a fusion of secretion tag and protein of interest
We used a “secretion tag” called OsmY. Synthesize a DNA sequence that contains a fusion of secretion tag and protein of interest
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iGEM – International Genetically EngineeredMachine
22 out of 34http://2014.igem.org/Team:Michigan
Modified ELISA allows us to test if our project works
Use electrophoresis to localize target protein in membrane -> use our excreted protein to bind target protein -> hit our our excreted protein with visualization antibodies
And…. It worked!
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DNA Sequencing
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Cost/Efficiency/Accuracy of DNA sequencing improving exponentially
Many ways to sequence DNA
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DNA Sequencing – Bead Imulsion
24 out of 34Mardis, E. Annu Rev Genomics Hum Genet 9, 387–402 (2008)
Different Companies have different sequencing methods – Roche/454 FLX was used to sequence James Watson, first human genome sequenced by next-gen sequencing
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25 out of 34Mardis, E. Annu Rev Genomics Hum Genet 9, 387–402 (2008)
DNA Sequencing – Sequencing by Synthesis
Illumina uses Sequencing by Synthesis TechnologyAgilent uses Sequencing by Ligation Technology
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DNA Synthesis – Artificial Life!
26 out of 34Gibson, D. et al. Science (2010).
Blue cells have chemically synthesized genome – contain an enzyme (β-galactosidase)
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DNA Synthesis – Artificial Life!
27 out of 34Gibson, D. et al. Science (2010).
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Genome Editing: CRISPR/Cas9
28 out of 34Charpentier, E. & Doudna, J. Nature (2013).
Wiedenheft, B. et al. Nature (2011).
CRISPR – Clustered Regularly Interspersed Short Palindromic RepeatsCas9 – CRISPR associated protein
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Genome Editing: CRISPR/Cas9Small Molecule Activation
29 out of 34Davis, K., Pattanayak, V., Thompson, D., Zuris, J. & Liu, D. Nat Chem Biol (2015).
CRISPR – Clustered Regularly Interspersed Short Palindromic RepeatsCas9 – CRISPR associated protein
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Engineering New Enzymes: Directed Evolution
30 out of 34Carbone, M. & Arnold, F. Current Opinion in Structural Biology (2007).
Protein Design Through Evolution and Homologous Recombination
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Computational Protein Design
31 out of 34Koga, N. et al. Nature (2012).
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Computational Protein Design
32 out of 34Koga, N. et al. Nature (2012).
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Virus Powered Batteries
33 out of 34Lee, Y. et al. Science (2009).
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Azotobacter vinelandii
Chlamydomonas reinhardtii
N2
Alternaria sp.
Cystathionine
Carbon
Synthetic Microbial Communities
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Thank you for your time!
Questions?