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Transcript of Attacking the Plastic Waste Problem: A Two …2008.igem.org/files/poster/Duke.pdfAttacking the...
Attacking the Plastic Waste Problem: A Two-Pronged ApproachKevin Chien, Lisa Zhang, Peter Zhu, Sam Wu, Sandy Sun, Vincent Ling
Faisal Reza, Zhongying Chen, Jingdong TianDuke University, Durham, North Carolina, 27708 USA
Goalspoly(3-hyroxybutyrate-co-4-hydroxybutyrate)
Introduction
Methods
Goals
Abstract
environmental pollution, a two-pronged approach with the po-
tential to solve these problems has been developed. Firstly, bio-
superior to petroleum-based plastics because they are both biode-
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polyethylene-degradation pathway is being engineered based on the
-dizes polyethylene and thereby increases its biodegradabil-
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petroleum-based plastics is a promising
MethodsPlastic
Catabolites
Plasmid Vector Name Vector PCR Reaction
Template for Insert Restriction enzymes
1 pASKphaCAB-noTag
pASK 1 phaCAB in PCR Blunt II Topo # 15
XbaIFw, BamHI
2 pASKphaCAB-tag pASK 2 phaCAB in PCR Blunt II Topo # 15
EcoRI, BamHI
3 pASKphaC-tag pASK 3 phaCAB in PCR Blunt II Topo # 15
EcoRI, BamHI
4 pSOSCat2-phaC pSOS 4 phaCAB in PCR Blunt II Topo # 15
BamHI, EcoRI
5 pASKphaAB-pLZCat2phaC
pASK 5 phaCAB in PCR Blunt II Topo # 15
XbaI, EcoRI,
6 pSOSCat2-phaC EcoRI, XhoI 6 pASKphaCAB-
pLZCat2 pASK 7 phaCAB in PCR
Blunt II Topo # 15 XbaI, EcoRI,
8 pSOSCat2-phaC #9 EcoRI, BamHI
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as the vector backbone. In the second method, Phusion enzyme was used
used as templates to create six di!erent vector constructs
Methods
Sequencing
NMR Analysis
Results
Fig.1 Colonies containing phaCAB insert glow red under UV light while those negative for the
insert do not appear red.
Fig.2 Close-up view of NMR of identifying region on commercial 3HB.
Fig.3 Close-up view of NMR of same identifying region on pASKphaCAB-noTag produced polymer.
Future Workcodon optimization
polyethylene-degradation pathway
Conclusion
Results
Acknowledgements
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desired mutant(s).
-termine region to mutate to allow LadA to accomodate polyethylene
inhibiting binding to longer substrates. The mutant library will be synthesized in a
Geobacillius thermodenitri!cans and test binding to a LadA native substrate: octadecane.
Fig.4 Computational docking of LadA to alkane substrates to determine interaction
energies of residues.
mtkkihinaf emncvghiah glwrhpenqr hrytdlnywt elaqllekgk fdalfladvv 60
giydvyrqsr dtavreavqi pvndplmlis amayvtkhla favtfsttye hpygharrms 120
tldhltkgri awnvvtshlp sadknfgikk ilehderydl adeylevcyk lwegswedna 180
virdienniy tdpskvhein hsgkyfevpg phlcepspqr tpviyqagms ergrefaakh 240 aecvflggkd vetlkffvdd irkrakkygr npdhikmfag icvivgkthd eameklnsfq 300
kywsleghla hygggtgydl skyssndyig sisvgeiinn mskldgkwfk lsvgtpkkva 360
demqylveea gidgfnlvqy vspgtfvdfi elvvpelqkr glyrvdyeeg tyreklfgkg 420
nyrlpddhia aryrnissnv 440
LadA Monooxygenase Amino Acid Sequence
Fig.5 Plot of interaction energies of residues versus alkane chain length. Length of bar denotes magnitude of interaction. Orange line separates native from non-native substrates and red box denotes residues we chose to mutate
Fig.6 Sequence of LadA from pWL1071 megaplasmid. The highlighted region is the region we plan to mutate. This region is a subregion of Insertion Region 4.
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to bind to longer substrates such as polyeth-ylene.
The wild-type LadA monooxygenase has also -
plasmid.
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polarity, size, and shape considerations yields
to acetoacetyl-CoA by "-ketothiolase, which is encoded by the phaA gene. This product is then re-
terminal oxidation upon polyethylene, a chemically inert and commercially prevalent plastic. Terminal
3 2 n 2 3n-alkane
LadA Monooxygenase
3 2 n 2 2primary alcohol
3 2 n 2
23 2 n
aldehyde
Alcohol Dehydrogenase
Aldehyde Dehydrogenase
NAD+
NAD+2