Attacking the ttac g t e Plastic Waste ast c aste Prob blem: - PowerPoint PPT Presentation

Attacking the ttac g t e Plastic Waste ast c aste Prob blem: A Two-Prong ed Approach Kevin Chien,Vincent Ling, Sandy Sun Sam Wu Lisa Sandy Sun, Sam Wu, Lisa Zhang, Peter Zhu

Introduction Introduction � Plastics have become a significant environment i ifi t i t tal t l Plastic waste problem � Devised two approache es to tackle this threat to tackle this threat Catabolites � Bioplastic synthesis � Plastic degradation

Bioplastic Synthe esis - Background � Polyhydroxyalkanoic acids ( PHA ) - natural storage polymer rs found in bacteria found in bacteria � Poly(3-hyroxybutyrate-co-4- hydroxybutyrate), or poly(3HB y y y ), p y( B- co-4HB) has elastic properties for wide range of applications � Pathways developed so far in E. P th d l d f i E . coli have yielded undesirably low w and unpredictable 4HB-to-3HB p ratios Approach 1 Goals Methods Results Discussion Background

Bioplastic Synth Bioplastic Synth hesis - Goals hesis - Goals � To engineer a controll ed biopathway for the biopathway for the production of poly(3HB B- co-4HB) 4HB) � To obtain more predictable compositio ons of the 4HB monomer i of the 4HB monomer i in in poly(3HB-co-4HB). Approach 1 Background Methods Results Discussion Goals

Bioplastic Synth Bioplastic Synth hesis- Methods hesis- Methods � 3HB Genetic Pathway � phaCAB operon genomic D DNA from Cupriavidus necator � Transformed and grown on n agar plates with Nile Red � 4HB Genetic Pathway 4HB Genetic Pathway � Cat2-phaC � pASK Constructs � pASK Constructs � Six different vector constru ucts in pASK created using PCR Blunt II phaCAB and PCR-Blunt II-phaCAB and pSOS-cat2 plasmids pSOS cat2 plasmids � Polymer extraction/cha aracterization by H1 NMR Approach 1 Background Goals Results Discussion Methods

Bioplastic Synth Bioplastic Synth hesis- Methods hesis- Methods Vector # Vector Insert Restriction Enzymes Construct pASKphaCAB- ASK h CAB pASK ASK 1 phaCAB in PCR Blunt II Topo # 15 (3.87kb) XbaIFw, BamHI noTag (2.92kb) pASK 2 pASKphaCAB-tag phaCAB in PCR Blunt II Topo # 15 (3.85kb) EcoRI, BamHI (3.0kb) (3.0kb) pASK 3 pASKphaC-tag phaCAB in PCR Blunt II Topo # 15 (1.77kb) EcoRI, BamHI (3.0kb) pSOS pSOS 4 pSOSCat2-phaC phaCAB in PCR Blunt II Topo # 15 (1.77kb) BamHI, EcoRI (3.99kb) pASKphaAB- pASK 1) phaCAB in PC CR Blunt II Topo # 1 (2.0kb) XbaI, EcoRI, EcoRI, 5 pLZCat2phaC (2.9kb) XhoI 2) pSOSCat2-pha aC (3.25 kb) 1) phaCAB in PC CR Blunt II Topo # 15 pASKphaCAB- pASK XbaI, EcoRI, EcoRI, 6 (3.86kb) pLZCat2 (2.9kb) BamHI 2) pSOSCat2-pha aC #9 (1.55kb) Approach 1 Background Goals Results Discussion Methods

Bioplastic Synth Bioplastic Synth hesis- Methods hesis- Methods tet promoter 3HB Construct X ba I (119 ) Tet repressor phaC pASKp phaCAB CamR 6 7 9 7 bp f1 origin phaA Ba m H I (39 9 7) phaB Approach 1 Background Goals Results Discussion Methods

Bioplastic Synth Bioplastic Synth hesis- Methods hesis- Methods 4HB Construct AmpR pUC19 UC19 C t2 Cat2 39 57 7 bp Cat2 LacZ promoter Approach 1 Background Goals Results Discussion Methods

Bioplastic Synth Bioplastic Synth hesis- Methods hesis- Methods Poly(3HB-co-4HB) tet promotor Construct Tet-repressor phaAB CamR pASK-phaAB-ter-p lacZCat2phaC 8 36 0 8 36 0 bp bp terminator f1 origin pLacZ Cat2 phaC Approach 1 Background Goals Results Discussion Methods

Bioplastic Synth Bioplastic Synth hesis - Results hesis - Results � Screening using Nile R Red indicator � Provides a visual indica ator of the presence of PHA of the presence of PHA A in A in E. coli � Problems: many coloni � Problems: many coloni es es fluoresce moderately without a strong signal without a strong signal Approach 1 Background Goals Methods Discussion Results

Bioplastic Synth Bioplastic Synth hesis- Results hesis- Results � Sequencing � Successful: pASKphaCA � Successful: pASKphaCA AB-noTag, pASKphaCAB-tag, AB-noTag pASKphaCAB-tag and pASKphaC were suc ccessfully constructed. � Mutation: pSOSCat-phaC � Mutation: pSOSCat-phaC C exhibited a frameshift C exhibited a frameshift mutation between cat2 a nd the lacZ promoter Approach 1 Background Goals Methods Discussion Results

Bioplastic Synth Bioplastic Synth hesis - Results hesis - Results � NMR of bioplastic NMR f bi l ti � Controls: � Commercial 3HB � Nonpolymer-producing ba acteria � NMR spectra for the nega NMR t f th ative control exhibit several ti t l hibit l peaks not present in comm mercial and pASKphaCAB polymers polymers � 5 ppm and 2.5 ppm area f for the commercial and pASKphaCAB noTag poly pASKphaCAB-noTag poly ymers are nearly identical ymers are nearly identical � E. coli transformed with p pASKphaCAB-noTag did produce poly(3HB) polym produce poly(3HB) polym mer mer Approach 1 Background Goals Methods Discussion Results

Bioplastic Synth Bioplastic Synth hesis - Results hesis - Results pASKphaCAB | Negative Control Approach 1 Background Goals Methods Discussion Results

Bioplastic Synth Bioplastic Synth hesis - Results hesis - Results � NMR of bioplastic NMR f bi l ti � Controls: � Commercial 3HB � Commercial 3HB � Nonpolymer-producing b bacteria � NMR spectra for the nega � NMR spectra for the nega ative control exhibit several ative control exhibit several peaks not present in com mmercial and pASKphaCAB polymers � 5 ppm and 2.5 ppm area fo r the commercial and pASKphaCAB-noTag polym mers are nearly identical � E. coli transformed with pAS � E coli transformed with pAS SKphaCAB noTag did produce SKphaCAB-noTag did produce poly(3HB) polymer Approach 1 Background Goals Methods Discussion Results

hesis - Results hesis - Results Discussion r producing control Results Methods Bioplastic Synth Bioplastic Synth NMR for non-polymer Goals Background Approach 1

hesis - Results hesis - Results Discussion Results mercial 3HB Methods Bioplastic Synth Bioplastic Synth NMR for comm Goals Background Approach 1

Bioplastic Synth Bioplastic Synth hesis - Results hesis - Results NMR for pASKphaCAB-n noTag produced polymer Approach 1 Background Goals Methods Discussion Results

Bioplastic Synth Bioplastic Synth hesis - Results hesis - Results � NMR of bioplastic � Controls: Controls: � Commercial 3HB � Nonpolymer-producing p y p g g g bacteria � NMR spectra for the neg gative control exhibit several peaks not present in com mmercial and pASKphaCAB polymers l � 5.3 ppm and 2.5 ppm a area for the commercial and pASKphaCAB polyme pASKphaCAB polyme rs are nearly identical rs are nearly identical � E. coli transformed wit th pASKphaCAB-noTag did produce poly(3HB) po produce poly(3HB) po lymer lymer Approach 1 Background Goals Methods Discussion Results

Bioplastic Synth Bioplastic Synth hesis - Results hesis - Results NMR for commercial 3HB at 2.5 ppm NMR for pASKphaCAB-noTag at 2.5 ppm Approach 1 Background Goals Methods Discussion Results

Bioplastic Synth Bioplastic Synth hesis - Results hesis - Results NMR for commercial 3HB at 5.3 ppm NMR for pASKphaCAB-noTag at 5.3 ppm Approach 1 Background Goals Methods Discussion Results

Bioplastic Synthe Bioplastic Synthe esis - Discussion esis - Discussion � Most of the plasmids have e been constructed and verified. � Need to finish constructio on of comprehensive plasmid containing the ph haCAB operon and the cat2phaC gene cat2phaC gene � Hein et. al. study produce ed poly(3HB-co-4HB) � Used recombinant plasmid U d bi t l id d d consisting of phaC and Cat2 i ti f h C d C t2 � Cultivation in the absence of glucose and the presence of 4-hydroxybutyrate 4 hydroxybutyrate � 4HB monomers were even ntually replaced by increasing amounts of 3HB, producin , p g p y( g poly(3HB-co-4HB). ) Approach 1 Background Goals Methods Results Discussion

Plastic Degradat Plastic Degradat tion tion � Biodegr adation of polyethylene � Polyet y hylene is chemically inert, and y y , accum mulating in landfill at a rate of almost t 25 million tons/year Approach 2 Goals Methods Results Discussion Background

Plastic Degradat Plastic Degradat tion: LadA tion: LadA Originally an alkane monooxygenase Shaped su uch that catalytic residues are located in deep pocket When doc cking alkane substrates, tail of alkanes lie e along Insertion Region 4 Requires cofactor FMN, which we e can remove to facilitate phage display assay. Separation of binding and catalyti ic residues mean that we can engineer binding without interferin engineer binding without interferin ng with activity ng with activity Approach 2 Goals Methods Results Discussion Background

Plastic Degradat Plastic Degradat tion tion � Goals: G l 1. Identify the mutable region of LadA 2. Synthesis large libra ry of LadA mutants with mutations in this regi mutations in this regi ion ion 3. Splice LadA mutants s into bacteriophages to conduct phage displa conduct phage displa ay assay ay assay. Approach 2 Background Methods Results Discussion Goals

Computational A Computational A Analysis Analysis LadA and highlighted LadA and highlighted D Docking alkanes with ki lk ith binding pocket 15 carbons to 63 carbons