- The document summarizes David Boyer's undergraduate research projects involving biofuels production through metabolic engineering of E. coli and recycling of algal waste. It also discusses current trends in biotechnology such as CRISPR genome editing, DNA sequencing/synthesis, and synthetic microbial communities. The research projects included engineering E. coli to tolerate high levels of isobutanol, recycling aqueous waste from algal hydrothermal liquefaction to grow more algae, and characterizing a cofactor and mechanism of a ferulic acid decarboxylase enzyme.

1. Undergraduate Research Summary
and Current State-of-the-Art in
Biotechnology
May 18, 2015
David Boyer
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2. 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

3. Introduction
Who am I? (Good Question)
Mona Shores High School – 2010
University of Michigan BS (Honors) Biochemistry and Chemistry – 2014
Ecovia 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, Biology
Email: davboyer@umich.edu
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4. 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

5. 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!

6. Isobutanol Tolerance Project
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Minty et al. Microb. Cell. Fact. 2011 10:18

7. Genome Evolution: Results
Mechanisms of adaptation
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Regulatory-stressresponseattenuation:
Minty et al. Microb. Cell. Fact. 2011 10:18
Evolution:MICincreasedfrom1%to2%w/v,144xmutationsacross6xlineages
Epistaticinteractions:Positiveepistasisbetweenhfqandsubsequentmutations

8. Genome Engineering
Overview
8 out of 34(George Church and Harris Wang)
Minty et al. Microb. Cell. Fact. 2011 10:18

9. Algae Biofuels - Overview
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10. Algae Hydrothermal Liquefaction:
Recycling Aqueous Waste
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11. 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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12. 12 out of 34
“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 in
stripped COMBO (no C, N, P) at various
concentrations of “raw” and “processed”
AqAl SBM
Algae Hydrothermal Liquefaction:
Recycling Aqueous Waste
Grow bacteria on Biofuels waste, feed it back to
algae!

13. 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
Recognition
Business 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

14. 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

15. 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)

16. 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

17. 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

18. 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).

19. Ferulic Acid Decarboxylase (FDC)
Structural Studies: X-ray Crystallography
19 out of 34Bhuiya et al. Applied and Environmental
Microbiology (2015).

20. iGEM – International Genetically Engineered
Machine
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!

21. iGEM – International Genetically Engineered
Machine
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

22. iGEM – International Genetically Engineered
Machine
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!

23. DNA Sequencing
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Cost/Efficiency/Accuracy of DNA sequencing
improving exponentially
Many ways to sequence DNA

24. 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

25. 25 out of 34Mardis, E. Annu Rev Genomics Hum Genet 9, 387–402 (2008)
DNA Sequencing – Sequencing by
Synthesis
Illumina uses Sequencing by Synthesis Technology
Agilent uses Sequencing by Ligation Technology

26. DNA Synthesis – Artificial Life!
26 out of 34Gibson, D. et al. Science (2010).
Blue cells have chemically synthesized genome
– contain an enzyme (β-galactosidase)

27. DNA Synthesis – Artificial Life!
27 out of 34Gibson, D. et al. Science (2010).

28. 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 Repeats
Cas9 – CRISPR associated protein

29. Genome Editing: CRISPR/Cas9
Small 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 Repeats
Cas9 – CRISPR associated protein

30. 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

31. Computational Protein Design
31 out of 34Koga, N. et al. Nature (2012).

32. Computational Protein Design
32 out of 34Koga, N. et al. Nature (2012).

33. 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

35. Thank you for your time!
Questions?