5-18-15 mona shores talk

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

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

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!

Isobutanol Tolerance Project

6 out of 34Minty et al. Microb. Cell. Fact. 2011 10:18

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

Genome EngineeringOverview

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

Algae Biofuels - Overview

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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


Grow bacteria on Biofuels waste, feed it back to algae!

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

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

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)

Ferulic Acid Decarboxylase (FDC)Kinetic and Mechanistic Studies


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



Hypothesized Mechanism

Computationally Predicted Mechanism

Reaction Energy Diagram

Can use computational chemistry software to help predict reaction mechanisms and transition state energies



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).

Ferulic Acid Decarboxylase (FDC)Structural Studies: X-ray Crystallography

19 out of 34Bhuiya et al. Applied and Environmental Microbiology (2015).

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!


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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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!

DNA Sequencing

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Cost/Efficiency/Accuracy of DNA sequencing improving exponentially

Many ways to sequence DNA

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 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

DNA Synthesis – Artificial Life!

26 out of 34Gibson, D. et al. Science (2010).

Blue cells have chemically synthesized genome – contain an enzyme (β-galactosidase)

DNA Synthesis – Artificial Life!

27 out of 34Gibson, D. et al. Science (2010).

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

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

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

Computational Protein Design

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

Computational Protein Design

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

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

Thank you for your time!

Questions?

5-18-15 mona shores talk

Documents

aqal aqal aqal sbm aqal

algae biofuels overview

algae inoculum

algae slurry

algae regrowth

sbm dilute sbm

biofuels waste

biofuels general overview