design of a microbial pretreatment for lignin degradation...
TRANSCRIPT
Dead Ligmen Tell No Tales Design of a Microbial Pretreatment for
Lignin Degradation using S. cerevisiae
Purdue iGEM 2015
Part One Motivation and Background
Initial Questions
Project Development
Water Sanitation Energy Building Agriculture
What are
Biodigesters?
• Definition: systems which decompose biological matter
using bacteria in an anaerobic environment in order to
produce a fuel source
• Four steps:
• Work best if separated
• One chamber bioreacters
Pretreatment Digestion Processing
And Filtration
Reuse and Disposal of
Waste
Why Biodigesters?
• Broad application
• Renewable energy
• Transportation
• Electricity generation
• Areas for improvement
Types of
Biodigestion
First Generation
Food plants
Second Generation
Fuel Crops
Third Generation
Algae
Fourth Generation
GM plants with less lignin
Lignin
• Nonrepeating
structure
• Protects plants
• Requires
pretreatment
Pretreatment
Thermal Mechanical
Chemical Enzymatic
Containment
• Usually rely on things from an industry setting
• But in less contained environments?
• Need a reliable, effective, affordable killswitch
Part Two Problem and Approach
Initial Problem
How can we design a simplified biological system
that covers all four stages of biodigestion?
Pretreatment
Disposal Processing
Digestion
J4 yeast Our system
Refined Problem
How can we use synthetic biology to
create a lower energy alternative to
thermal pretreatment?
How can we contain such a system in different
settings including third world application?
Solution
Cellulose
Lignin
Enzyme Cocktail
Our System
J4 Yeast
Enzyme Selection
Aldo-Keto Reductase Lignin Peroxidase Laccase
Tyrosinase Manganese Peroxidase Versatile Peroxidase
Genetic Construct
Design
Biobrick prefix
Kozack Sequence
Biobrick suffix
Manganese Peroxidase Fusion Terminator
yEGFP
Constitutive Promoter
Linker Linker
Manganese Peroxidase Secretion Tag
Killswitch
Considerations
• Several promoters considered
• Robust killswitch
• Ethanol based spill kit
Biobrick prefix
Kozack Sequence
Biobrick suffix
Lambda Holin Coding Sequence Terminator Negative Promoter
Plasmid Optimization
Codon
Optimization
Refinement
for blocks
gBlock
Format
Restriction
Site Check
Part Three Methods and Results
One Project,
Two Teams
Enzyme Team
• Suraj, Jill, Erich, and Melissa
• Construction of enzyme constructs and ligation into yeast
vector
Killswitch Team
• Bowman, Kate, and Alexa
• Construction of oxygen based killswitch
Enzyme and Killswitch Track
Methodology
Enzyme Track
Progress
Enzyme Track
Problems
• Gibson was not able to:
• Combine the G-blocks at
proper site
• Produce DNA at ample
concentrations for
transformation
• Attempted Overlap PCR to
alleviate issues
• Limited success with
Manganese Peroxidase
LiP VP AKR MnP
Assays
• Completed
• MTT Assay • Yeast Growth/Death Curves
• Future Experiments
• Bradford Protein Assay • Protein concentration measurement
• Peroxidase Assay • Lignin, Manganese, and Versatile Peroxidase activity
• DMP Assay • Laccase activity assay
• Klason Procedure • Lignin Degradation Assay
Overall Progress
Enzyme PCR Amp
Purify/Extract
Gibson E.coli Vector Insert
E.coli Transformation
Miniprep Sequencing Yeast Transformation
Lignin Peroxidase
Laccase
Manganese Peroxidase
AKR
Versatile Peroxidase
Tyrosinase
Completed Stopped
Enzyme Analysis
• Since enzymes are exported from cells, only supernatant needs to be
analyzed
• Enzymes would then be tested in multiple combinations
• Linear regression model used to calculate lignin breakdown of each
enzyme
• Statistical optimization used to eliminate combinations with little effect and
determine amount of enzymes needed for bioreactor
Conclusions
• Unable to reach proof of concept point
• Errors:
• Overlapping restriction site error
• block design errors
• Still believe the idea is viable
• Better gene design in the future
Part Four Human Practices and Beyond
Changing Human
Practices Focus
• Started broad
• Shift to pretreatment
• Biofuel application
• Broad potential
Enzymatic Pretreatment B
road
Bro
ad
Biofuels
Cardinal Ethanol
Plant
• Full time ethanol plant
• 100 million gal / year
• Ethanol is sold to gasoline companies
Nonscientific Issues
Surrounding Biofuels
• Lignocellulosic biofuels only
• Transportation infrastructure
• Cost
• All biofuels
• Cannot be shipped in current pipelines
• Cannot be used in most current vehicles
• Current gasoline market
Viability
So is any of this even worth it?
We don’t know yet.
But…
Assay data would have helped build a model.
Model would have been used to predict overall costs and
evaluate how much is being saved.
Final Thoughts
• Lowered cost opens doors
• Standardized platform
• Wide spectrum of application
Summary
• Examined problems facing biodigesters
• Surveyed range of lignin-degrading enzymes
• Selected candidate enzymes based on design criteria
• Designed genetic constructs for expression
• Had constructs synthesized and assembled four of the devices
• Designed oxygen-dependent kill switch
• Constructed kill switch using registry parts
• Visited Cardinal Ethanol Plant
• Used real world perspective to develop standards of feasibility
Attributions
• Advisors
– Professor Jenna Rickus
– Soo Ha
– Sam Lee
– Janie Brennan
– Professor Michael Gribskov
• Project consultation
– Professor Michael Scharf
– Professor Nathan Mosier
• Collaboration
– Cardinal Ethanol Plant
– Purdue Society of Women
Engineers
Funding
• Purdue University Office of the Provost
• Purdue University Office of the Vice
President of Research
• Purdue University College of Agriculture
• Purdue University College of Science
• Purdue University College of Engineering
• Purdue University Honors College
• Purdue University Department of Agricultural
and Biological Engineering
• Purdue University Learning Beyond the
Classroom Grant
• Purdue University Summer Undergraduate
Research Fellowship Program
• Purdue University Molecular Agriculture
Summer Institutes Program
• Purdue University Day of Giving
Thank You Questions?
Background
&
Motivation
Problem
&
Approach
Methods
&
Results
Human Practices
&
Beyond