make it or break it - 2011.igem.org2011.igem.org/files/presentation/washington.pdf · make it or...
TRANSCRIPT
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Make it or Break it: Diesel Production
and Gluten Destruction the Synthetic Biology Way
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Washington iGEM 2011 Objectives
Diesel Production
Gluten Destruction
iGEM Toolkits
Community Outreach
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Making it: work on diesel production
Diesel Production
Gluten Destruction
iGEM Toolkits
Community Outreach
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Society is dependent upon limited petroleum reserves
Combustion
CO2
Marine
Flight
Long Distance Trucking
Petroleum Reserves Alkanes
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Integrating CO2 into fuel production
CO2
Combustion
Petroleum Reserves
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Biofuel Production Agricultural Products
Combustion
Carbon Dioxide
OH Alcohols FAMEs
Biofuels are renewable, yet also inefficient, incompatible, and corrosive
( )n
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An ideal fuel is both renewable and efficient
Petroleum Biofuel
R E N E W A B I L I T Y E N E R G Y D E N S I T Y
Microbially-Produced Alkanes
?
OH
C O M P A T I B I L I T Y
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O
S-ACP
O
H
Schirmer, A., M. A. Rude, et al. "Microbial Biosynthesis of Alkanes." Science 329.5991 (2010): 559-62.
Microbial production of alkanes from fatty acids
Cellular Fatty Acid Biosynthesis
Aldehyde DeCarbonylase
Acyl-ACP Reductase
AAR
ADC
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The basic alkane production pathway
Strong constitutive
promoter RBS ADC AAR RBS
Bba_K590025
Bba_K590026 Bba_K590031 Bba_K590032
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Analyzing alkane production with GCMS
To GCMS
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To GCMS
Analyzing alkane production with GCMS
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Analyzing alkane production with GCMS
Average of 9 .287 to 9.316 m in.: 71.D \data.m s PentadecaneH ead to T ail M F =764 R M F =909
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
0
50
100
50
100
30
43
43
57
57
61
71
71
85
85
99
99
113
113 126
127 140
141
155
155
169
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183
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193 202212
212
221
Gas Chromatograph Mass Spectrometer
Experimental Spectrum
Reference Spectrum
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ADC expression alone is not sufficient for hydrocarbon production
Rel
ativ
e ab
un
dan
ce
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AAR expression results in production of a C14 alcohol
C14 Alcohol
Rel
ativ
e ab
un
dan
ce
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Alkane production from the PetroBrick
C13 Alkane
C15 Alkane C14 Alcohol &
C17 Alkene
Rel
ativ
e ab
un
dan
ce
We converted sugar into diesel!
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Initial alkane yield
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Optimized alkane yield
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System modifications
Decarbonylase Redesign
Alternative Chassis
Enzyme Localization
System Optimization
Branched Alkanes
Alternative Aldehyde
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From making it to breaking it: gluten destruction
Diesel Production
Gluten Destruction
iGEM Toolkits
Community Outreach
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Gluten intolerance is a common and difficult problem
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Indigestible gluten peptides trigger an immune response
Gluten P L
Q P
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Indigestible gluten peptides trigger an immune response
Gluten P L
Q P
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A protein therapeutic is in clinical trials… but is has low activity at pH 4
• SC PEP from Sphingomonas capsulata
• Low activity in acidic conditions
• Good activity on PQLP
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Kumamolisin is optimal at pH 4… but it has unknown activity on PQLP
• Endopeptidase from Alicyclobacillus sendaiensis
• High activity in acidic conditions
• Unknown activity on PQLP
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An ideal treatment is optimal at low pH and has PQLP activity
Activity on PQLP Activity at pH 4 Ideal
Treatment
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We used computational tools to redesign Kumamolisin for PQLP
Native active site
G319S variant
Mutate
To download your copy of Foldit, go to http://Fold.It
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To test our designs, we developed a whole cell lysate assay
P Q
P L Q P
L P
P L Q P
Over 100 novel mutants tested!
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Activity of mutants from whole cell lysate screen
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The top mutants were purified and characterized: over 10-fold improvement
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Greater than 100-fold improvement with a combinatorial mutant
118x activity
1x activity 0.15x activity
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UW iGEM Objectives
Diesel Production
Gluten Destruction
iGEM Toolkits
Community Outreach
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Gibson Assembly Toolkit
Gibson Assembly
E N X Insert S N P
Standard Biobrick Vectors RFC 10
S1 S2
Gibson Assembly Vectors RFC 21 (BglBrick)
…GCGGCCGC... ...CGCCGGCG…
Bg B
a X
…CTGCTGCGGAGATCT… …AACAGGGTTCTCGAG…
plasmids 1A3 1C3 3K3 4A5 4C5
SB SB SB SB SB SB
GA GA GA GA GA GA
p p p p p p
“plasmids for Gibson Assembly”
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Improved cloning efficiency with new pGA vectors
pSB Vector Cloning
pGA Vector Cloning
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Gibson Assembly Efficiencies
pSB pGA
Effi
cie
ncy
12%
99%
GFP
pGA1A3
GFP
pSB1A3
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Goal: Build magnetic E. coli – “MagnetoColi”
Magnetotactic Bacteria
Extracted 18 genes from mamAB operon that encode scaffold polymerization, vesicle formation, and biomineralization*
Magnetosome Toolkit
*Thanks to the Komeili lab (Berkeley) for the genomic DNA! (Magnetospirillum magneticum, AMB-1)
mamH I E J K L M N O P A Q R B S T U V
E.coli
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Magnetosome Toolkit gene characterization
sfGFP-MamK scaffold polymerization
MamK/I sfGFP Promoter RBS
Membrane localized sfGFP-MamI, binds MamK
+
Full Characterization & Assembly
MagnetoColi!
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UW iGEM 2011 Objectives
Diesel Production
Gluten Destruction
iGEM Toolkits
Community Outreach
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Showing synthetic biology to the community!
Engineering Discovery Days
Bennett Elementary
School
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Teaching the community about cloning!
Cut
Balloon Cell Ribbon Vector
Linearized Plasmid DNA Insert
Ligate Transform
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Teaching the community about cloning!
Completed Cell with New Vector!
Linearized Plasmid
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What We Accomplished
Diesel Production
Gluten Destruction
iGEM Toolkit
Community Outreach
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What We Accomplished
Made diesel from sugar
Engineered enzyme to improve activity 100 fold
Gibson Assembly Toolkit
Magnetosome Toolkit
Showed community synthetic biology is awesome
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G319S, 358G, D368H
Biobricks Submitted
Kumamolisin-As
Gluten Destruction
5
MamSTU iGEM Toolkits
21
Petrobrick
Diesel Production
39
Total Biobricks
65!!!
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Alicia Wong Angus Toland Austin Moon Benjamin Mo
Casey Ager Chris Choe Cindy Wu
Daniel Hadidi David Zong Elaine Lai
iGEM Students
Acknowledgments
iGEM Advisors
Aaron Chevalier Alan Weiner Chris Eiben David Baker
Dominic Chung Eric Klavins
Ingrid Swanson Pultz Jeremy Mills Justin Siegel Ling Lin Liu Matt Smith Rob Egbert
Juhye An Lei Zheng Liz Stanley
Marika Cheng Matthew Harger Michael Brasino
Rashmi Ravichandran Sarah Wolf Sean Wu
Seth Sagulo Sydney Gordon
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Questions??