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-

-

polyethylene-degradation pathway is being engineered based on the

-dizes polyethylene and thereby increases its biodegradabil-

-

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

-

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

-

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.

-

-

to bind to longer substrates such as polyeth-ylene.

The wild-type LadA monooxygenase has also -

plasmid.

-

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