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

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

Dec 18, 2022 226 likes •588 views

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

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SLIDE 1

Attacking the ttac g t e Prob

A Two-Prong

Plastic Waste ast c aste blem:

ed Approach

Kevin Chien,Vincent Ling, Sandy Sun Sam Wu Lisa Sandy Sun, Sam Wu, Lisa Zhang, Peter Zhu

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SLIDE 2

Introduction Introduction

Plastics have become

i ifi t i t significant environment waste problem

Devised two approache

to tackle this threat to tackle this threat

Bioplastic synthesis Plastic degradation

a t l tal

Plastic

es

Catabolites

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SLIDE 3

Bioplastic Synthe Background

Polyhydroxyalkanoic acids

(PHA) - natural storage polymer found in bacteria found in bacteria

Poly(3-hyroxybutyrate-co-4-

hydroxybutyrate), or poly(3HB y y y ), p y( co-4HB) has elastic properties for wide range of applications P th d l d f i E

Pathways developed so far in E.

coli have yielded undesirably low and unpredictable 4HB-to-3HB p ratios

Background Goals

Approach 1

esis -

rs B- . w

Methods Results Discussion

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SLIDE 4

Bioplastic Synth Bioplastic Synth

To engineer a controll

biopathway for the biopathway for the production of poly(3HB 4HB) co-4HB)

To obtain more

predictable compositio

  • f the 4HB monomer i
  • f the 4HB monomer i

poly(3HB-co-4HB).

Background Goals

Approach 1

hesis - Goals hesis - Goals

ed B-

  • ns

in in

Methods Results Discussion

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SLIDE 5

Bioplastic Synth Bioplastic Synth

3HB Genetic Pathway

phaCAB operon genomic D Transformed and grown on

4HB Genetic Pathway

4HB Genetic Pathway

Cat2-phaC

pASK Constructs pASK Constructs

Six different vector constru

PCR Blunt II phaCAB and PCR-Blunt II-phaCAB and

Polymer extraction/cha

Background Goals

Approach 1

hesis- Methods hesis- Methods

DNA from Cupriavidus necator n agar plates with Nile Red ucts in pASK created using pSOS cat2 plasmids pSOS-cat2 plasmids

aracterization by H1 NMR

Methods Results Discussion

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SLIDE 6

Bioplastic Synth Bioplastic Synth

# Vector Construct Vector

ASK h CAB ASK 1 pASKphaCAB- noTag pASK (2.92kb) phaCAB in PCR 2 pASKphaCAB-tag pASK (3.0kb) phaCAB in PCR (3.0kb) 3 pASKphaC-tag pASK (3.0kb) phaCAB in PCR pSOS 4 pSOSCat2-phaC pSOS (3.99kb) phaCAB in PCR 5 pASKphaAB- pLZCat2phaC pASK (2.9kb) 1) phaCAB in PC 2) pSOSCat2-pha 6 pASKphaCAB- pLZCat2 pASK (2.9kb) 1) phaCAB in PC (3.86kb) 2) pSOSCat2-pha Background Goals

Approach 1

hesis- Methods hesis- Methods

Insert Restriction Enzymes

Blunt II Topo # 15 (3.87kb) XbaIFw, BamHI Blunt II Topo # 15 (3.85kb) EcoRI, BamHI Blunt II Topo # 15 (1.77kb) EcoRI, BamHI Blunt II Topo # 15 (1.77kb) BamHI, EcoRI CR Blunt II Topo # 1 (2.0kb) aC (3.25 kb) XbaI, EcoRI, EcoRI, XhoI CR Blunt II Topo # 15 aC #9 (1.55kb) XbaI, EcoRI, EcoRI, BamHI Methods Results Discussion

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SLIDE 7

Bioplastic Synth Bioplastic Synth

3HB Construct

Tet repressor

pASKp

6 7

CamR f1 origin phaB

Ba mH I (39 9 7)

Background Goals

Approach 1

hesis- Methods hesis- Methods

tet promoter

X baI (119 )

phaC

phaCAB

9 7 bp

phaA

Methods Results Discussion

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SLIDE 8

Bioplastic Synth Bioplastic Synth

4HB Construct

UC19 pUC19

39 57

Cat2 LacZ promoter

Background Goals

Approach 1

hesis- Methods hesis- Methods

AmpR

C t2 Cat2

7 bp

Methods Results Discussion

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SLIDE 9

Bioplastic Synth Bioplastic Synth

Poly(3HB-co-4HB) Construct

Tet-repressor

pASK-phaAB-ter-p

8 36 0

CamR

8 36 0

f1 origin

Background Goals

Approach 1

phaC

hesis- Methods hesis- Methods

tet promotor phaAB

lacZCat2phaC

bp bp

pLacZ terminator Cat2

Methods Results Discussion

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SLIDE 10

Bioplastic Synth Bioplastic Synth

Screening using Nile R

indicator

Provides a visual indica

  • f the presence of PHA
  • f the presence of PHA
  • E. coli

Problems: many coloni Problems: many coloni

fluoresce moderately without a strong signal without a strong signal

Background Goals

Approach 1

hesis - Results hesis - Results

Red

ator A in A in es es

Methods Results Discussion

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SLIDE 11

Bioplastic Synth Bioplastic Synth

Sequencing

Successful: pASKphaCA Successful: pASKphaCA

and pASKphaC were suc

Mutation: pSOSCat-phaC Mutation: pSOSCat-phaC

mutation between cat2 a

Background Goals

Approach 1

hesis- Results hesis- Results

AB-noTag pASKphaCAB-tag AB-noTag, pASKphaCAB-tag, ccessfully constructed. C exhibited a frameshift C exhibited a frameshift nd the lacZ promoter

Methods Results Discussion

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SLIDE 12

Bioplastic Synth Bioplastic Synth

NMR f bi l ti

NMR of bioplastic

Controls:

Commercial 3HB Nonpolymer-producing ba

NMR t f th

NMR spectra for the nega

peaks not present in comm polymers polymers

5 ppm and 2.5 ppm area f

pASKphaCAB-noTag poly pASKphaCAB noTag poly

  • E. coli transformed with p

produce poly(3HB) polym produce poly(3HB) polym

Background Goals

Approach 1

hesis - Results hesis - Results

acteria

ti t l hibit l ative control exhibit several mercial and pASKphaCAB for the commercial and ymers are nearly identical ymers are nearly identical

pASKphaCAB-noTag did mer mer

Methods Results Discussion

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SLIDE 13

Bioplastic Synth Bioplastic Synth

pASKphaCAB |

Background Goals

Approach 1

hesis - Results hesis - Results

Negative Control

Methods Results Discussion

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SLIDE 14

Bioplastic Synth Bioplastic Synth

NMR f bi l ti

NMR of bioplastic Controls: Commercial 3HB Commercial 3HB Nonpolymer-producing b NMR spectra for the nega NMR spectra for the nega

peaks not present in com polymers

5 ppm and 2.5 ppm area fo

pASKphaCAB-noTag polym

E coli transformed with pAS

  • E. coli transformed with pAS

poly(3HB) polymer

Background Goals

Approach 1

hesis - Results hesis - Results

bacteria ative control exhibit several ative control exhibit several mmercial and pASKphaCAB r the commercial and mers are nearly identical SKphaCAB noTag did produce SKphaCAB-noTag did produce

Methods Results Discussion

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SLIDE 15

Bioplastic Synth Bioplastic Synth

NMR for non-polymer

Background Goals

Approach 1

hesis - Results hesis - Results

r producing control

Methods Results Discussion

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SLIDE 16

Bioplastic Synth Bioplastic Synth

NMR for comm

Background Goals

Approach 1

hesis - Results hesis - Results

mercial 3HB

Methods Results Discussion

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SLIDE 17

Bioplastic Synth Bioplastic Synth

NMR for pASKphaCAB-n

Background Goals

Approach 1

hesis - Results hesis - Results

noTag produced polymer

Methods Results Discussion

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SLIDE 18

Bioplastic Synth Bioplastic Synth

NMR of bioplastic

Controls:

Controls:

Commercial 3HB Nonpolymer-producing

p y p g

NMR spectra for the neg

peaks not present in com l polymers

5.3 ppm and 2.5 ppm a

pASKphaCAB polyme pASKphaCAB polyme

  • E. coli transformed wit

produce poly(3HB) po produce poly(3HB) po

Background Goals

Approach 1

hesis - Results hesis - Results

g bacteria g

gative control exhibit several mmercial and pASKphaCAB area for the commercial and rs are nearly identical rs are nearly identical th pASKphaCAB-noTag did lymer lymer

Methods Results Discussion

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SLIDE 19

Bioplastic Synth Bioplastic Synth

NMR for commercial 3HB at 2.5 ppm

Background Goals

Approach 1

hesis - Results hesis - Results

NMR for pASKphaCAB-noTag at 2.5 ppm

Methods Results Discussion

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SLIDE 20

Bioplastic Synth Bioplastic Synth

NMR for commercial 3HB at 5.3 ppm

Background Goals

Approach 1

hesis - Results hesis - Results

NMR for pASKphaCAB-noTag at 5.3 ppm

Methods Results Discussion

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SLIDE 21

Bioplastic Synthe Bioplastic Synthe

Most of the plasmids have

verified.

Need to finish constructio

plasmid containing the ph cat2phaC gene cat2phaC gene

Hein et. al. study produce

U d bi t l id

Used recombinant plasmid Cultivation in the absence

4 hydroxybutyrate 4-hydroxybutyrate

4HB monomers were even

amounts of 3HB, producin , p

Background Goals

Approach 1

esis - Discussion esis - Discussion

e been constructed and

  • n of comprehensive

haCAB operon and the ed poly(3HB-co-4HB)

d i ti f h C d C t2 d consisting of phaC and Cat2

  • f glucose and the presence of

ntually replaced by increasing g poly(3HB-co-4HB). g p y( )

Methods Results Discussion

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SLIDE 22

Plastic Degradat Plastic Degradat

Biodegr

Polyet

y accum almost

Background Goals

Approach 2

tion tion

adation of polyethylene

hylene is chemically inert, and y y , mulating in landfill at a rate of t 25 million tons/year

Methods Results Discussion

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SLIDE 23

Plastic Degradat Plastic Degradat

Originally Shaped su located in When doc alkanes lie Requires cofactor FMN, which we display assay. Separation of binding and catalyti engineer binding without interferin

Background Goals

Approach 2

engineer binding without interferin

tion: LadA tion: LadA

an alkane monooxygenase uch that catalytic residues are deep pocket cking alkane substrates, tail of e along Insertion Region 4 e can remove to facilitate phage ic residues mean that we can ng with activity

Methods Results Discussion

ng with activity

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SLIDE 24

Plastic Degradat Plastic Degradat

G l

Goals:

  • 1. Identify the mutable
  • 2. Synthesis large libra

mutations in this regi mutations in this regi

  • 3. Splice LadA mutants

conduct phage displa conduct phage displa

Background Goals

Approach 2

tion tion

region of LadA ry of LadA mutants with ion ion s into bacteriophages to ay assay ay assay.

Methods Results Discussion

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SLIDE 25

Computational A Computational A

LadA and highlighted LadA and highlighted binding pocket

Analysis Analysis

D ki lk ith Docking alkanes with 15 carbons to 63 carbons

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SLIDE 26

C15 C18 C20 C21 C24 C25 C27 C30 C33 C Phe 10 Met 12 Thr 14 i 1

Native Substrates

His 17 Ile 18 Phe 55 Ala 57 Asp 58 Val 59 Tyr 63 Val 65 Gln 79 Thr 104 Asn 133 Thr 136 Ser 137 His 138 Leu 139 Pro 140 Ile 151 His 154 Tyr 158 Ala 227 Gly 228 Met 229 Ser 230 Phe 245 e 5 Gly 247 Gly 248 Lys 249 Asp 250 Glu 252 Thr 253 Phe 256 Phe 257 Phe 257 Trp 303 His 308 His 311 Tyr 312 Gly 313 Gly 314 Gly 315 Thr 316 Thr 316 Tyr 318 Ile 329 Gly 330 Ser 331 Ile 332 Glu 336 Ile 337 l Ile 338 Asn 340 Met 341 Asp 345 Asp 345 Lys 347 Trp 348 Tyr 365 36 C39 C40 C42 C45 C48 C51 C54 C57 C60 C63

Non-native Substrates

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SLIDE 27

Plastic Degradat Plastic Degradat

i i Interaction Energies Native Substrates

Other IS4 44% Other 25% 44% Inner Inner Pocket 31%

Background Goals

Approach 2

ion - Results ion Results

i i Interaction Energies Non-native substrates

IS4 39% Other 38% Inner Pocket 21%

Methods Results Discussion

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SLIDE 28

Plastic Degradat Plastic Degradat

C15 C18 C20 C21 C24 C25 C27 C30 C33 C36 C39 C40 C42 C45 C48 C51 C54 C57 C Phe 10 Met 12 Thr 14 His 17 Ile 18 Phe 55 Ala 57 Asp 58 Val 59 Tyr 63 Val 65 Val 65 Gln 79 Thr 104 Asn 133 Thr 136 Ser 137 His 138 Leu 139 Pro 140 Ile 151 His 154 Tyr 158 Ala 227 Gly 228 Met 229 Ser 230 Phe 245 Gly 247 Gly 248 Lys 249 Asp 250 Glu 252 Thr 253 Phe 256 Phe 256 Phe 257 Trp 303 His 308 His 311 Tyr 312 Gly 313 Gly 314 Gly 315 Thr 316 Tyr 318 Ile 329 Gly 330 Ser 331 Ile 332 Glu 336 Ile 337 Ile 338 Asn 340 Met 341 Asp 345 Asp 345 Lys 347 Trp 348 Trp 348 Tyr 365

Background Goals

Approach 2

tion - Results tion - Results

C60 C63

The region outlined in the red box is a subregion of Insertions Region 4 and Insertions Region 4 and contains the residues we have identified for mutation.

Methods Results Discussion

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SLIDE 29

LadA Sequence region

10 20 30 MTKKIHINAF EMNCVGHIAH GLWRHPENQR HRY 70 80 90 GIYDVYRQSR DTAVREAVQI PVNDPLMLIS AMA 130 140 150 TLDHLTKGRI AWNVVTSHLP SADKNFGIKK ILE TLDHLTKGRI AWNVVTSHLP SADKNFGIKK ILE 190 200 210 VIRDIENNIY TDPSKVHEIN HSGKYFEVPG PHL 250 260 270 AECVFLGGKD VETLKFFVDD IRKRAKKYGR NPD 310 320 330 KYWSLEGHLA HYGGGTGYDL SKYSSNDYIG SIS 370 380 390 DEMQYLVEEA GIDGFNLVQY VSPGTFVDFI ELV 430 440 NYRLPDDHIA ARYRNISSNV

+ Mutable

40 50 60 YTDLNYWT ELAQLLEKGK FDALFLADVV 100 110 120 AYVTKHLA FAVTFSTTYE HPYGHARRMS 160 170 180 EHDERYDL ADEYLEVCYK LWEGSWEDNA EHDERYDL ADEYLEVCYK LWEGSWEDNA 220 230 240 LCEPSPQR TPVIYQAGMS ERGREFAAKH 280 290 300 DHIKMFAG ICVIVGKTHD EAMEKLNSFQ 340 350 360 SVGEIINN MSKLDGKWFK LSVGTPKKVA 400 410 420 VVPELQKR GLYRVDYEEG TYREKLFGKG

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SLIDE 30

Plastic Degradat Plastic Degradat

Replaced three residues (Isoleucine 33 m tagenesis i ard The replace mutagenesis wizard. The replace considerations of size, shape, and p fold increase in binding affinity to no g y

Background Goals

Approach 2

tion- Discussion tion- Discussion

37, Asparagine 340, Tryptophan 348) in PyMOL

ements ere selected ith the ements were selected with the

  • polarity. This mutant showed a six-
  • n-native substrates.

Methods Results Discussion

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SLIDE 31

Experimental Experimental

We have also extracte

designed primers to P designed primers to P the control run, . L dA t t lib i

LadA mutant library is

mentor’s oligonucleoti ed the genome and CR wild-type LadA for CR wild type LadA for th i d i synthesized using our de synthesizer

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SLIDE 32

Bioplastic Synth Future Direction

Optimize the ratio of 4

in poly(3HB-co-4HB).

Site-directed mutag Codon optimization Codon optimization Better screening me

b t 3HB d 4 between 3HB and 4

Using lipases, which

monomers, to degrad monitor the lipase act

hesis - n

4HB to 3HB monomers enesis ethods for the ratio 4HB i th l 4HB in the copolymer

  • nly degrade 4HB

de the plastic and then tivity

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SLIDE 33

Plastic Degrada Direction

Perform multiple round Codon-optimize desire Codon optimize desire

expression in E.coli. C bi L dA i h h

Combine LadA with oth

polyethylene-degradat complete system.

ation- Future

ds of directed evolution. ed mutants for ed mutants for h f her components of ion pathway to form a

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SLIDE 34

Final Remarks Final Remarks

Increase bioplastic pro Obtain more desirable Obtain more desirable

properties

Biodegrade plastics on

biodegradable g

  • duction efficiency

e bioplastic elasticity e bioplastic elasticity nce thought to be non-

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SLIDE 35

Acknowledgeme Acknowledgeme

iGEM Zhongying Chen Zhongying Chen Faisel Reza

  • Dr. Myra Halpin

Dr Amy Sheck

  • Dr. Amy Sheck
  • Dr. Jingdong Tian

ents ents