Chew Fight Marseille (France) Sao Paolo (Brasil) Xiamen (China) - - PowerPoint PPT Presentation

Chew Fight

Team Aix-Marseille 2015

Team Aix-Marseille 2015

Xiamen (China) Georgia (USA) Marseille (France) Sao Paolo (Brasil) Montevideo (Uruguay)

374 billion

• f chewing gum

sticks are made every year

100,000 tons

• f chewing gum are

consumed each year

Did you know ?

Did you know ?

Decomposed in 5 years Dangerous for the environment and animals Cities spend millions of dollars to clean it Waste of time, energy and water

Our project : To degrade bubble gum

Bubble gum composition

Polystyrene butadiene Polyisoprene

CH2 CH2 C CH CH3 CH2 CH CH2 CH CH CH2

Gum base 60% Sugar 20% Syrup 20%

n n

Sato, Shin, et al. "Degradation of vulcanized and nonvulcanized polyisoprene rubbers by lipid peroxidation catalyzed by oxidative enzymes and transition metals." Biomacromolecules 4.2 (2003): 321-329

Latex clearing protein

• Specific metal oxidase

Which enzymes ?

Which enzymes ?

• General metal oxidase

Laccase CuII CuI H+ +

𝟐 𝟓O2

Ruthenium

Schneider, Ludovic, et al. "Visible‐Light‐Driven Oxidation of Organic Substrates with Dioxygen Mediated by a [Ru (bpy) 3] 2+/Laccase System."ChemSusChem (2015)

• Oxydase

Laccase CuII CuI H+ +

𝟐 𝟓O2

• Hydroxylase

Cytochrome C FeII FeIII

Our solution

Oxidised cyt C * FeIII* Very oxidised Cyt C FeIV Oxydised Laccase CuII Oxidised laccase CuII Reduced laccase CuI Reduced Laccase CuI H+ +

R R .+ O2 R O H+ +

Oxidised Cyt C FeIII Reduced Cyt C FeII hv

Proposition

Laccase can oxidize Cytochrome C

Abs (550 nm)

Proof of concept

Our constructions

pT7 = T7 promoter RBS = Ribosome binding site LCP = Latex clearing protein

pT7 - RBS Laccase Cytochrome C His pT7 - RBS LCP His pT7 - RBS His pT7 - RBS Laccase Linker Cytochrome C His

• G. polyisoprenivorans

Streptomyces sp K30

• E. coli
• T. thermophilus
• B. Subtilis
• P. denitrificans
• S. oneidensis

Synechosystis PCC 6803

Modeling

Cytochrome C Laccase Linker Using linker generator 1.0 Made by Wilson Goma Laccase (T. thermophilus) Cytochrome C (P. denitrificans) Linker 9 linkers P T7 - RBS Laccase Linker Cytochrome C His

Laccase results

P T7 - RBS Laccase His kDa IPTG kDa Ladder

• E. coli

Western blot revealed by antibody against His- tag SDS-PAGE Comassie brillant blue

3

Chassis BL21 (DE3) Purification

Laccase activity

[O2] (%) Time (s)

Laccase from ISM2 ABTS without laccase Our laccase with ABTS Our laccase incubated 20 min with 100 mM copper

Cytochrome C results

1- Ladder 2- Cytoplasm 3- Solubilized membranes

Cytochrome C His P T7 - RBS

• S. oneidensis

Western blot revealed by antibody against His-tag

Next step…

Human practices

• Antonio Lopez (1869) : Arrived in New York with the first bubble gum
• William Semple (1823) : Patent to create good taste
• William White (1895) : Glucose syrup into basic gum
• William Wrigley Jr (1932) : Launched bubble gum in the USA

W.S W.W W.W Jr A.L

Celibrities

Next celibrities ?

New cleaning device :

• 1. Spray the enzymes
• 2. Scrape them off

 Has to work quickly

Applications

Patch:

• 1. Put it on the ground
• 2. Wait as long as it’s needed
• 3. Remove the patch

 Can stay longer & work better

Applications

Our product

• Eco-responsible
• Saves Water
• Saves Energy
• Saves Money
• No chemical products
• Major issue : solution based on GMOs

30% 20% 50% Rather against (1 to 4) Neutral (5) Rather for (6 to 10)

People’s opinion

« On a scale of 1 to 10, are you against or for GMOs ? » In total we had 584 replies from all around the world.

Communication campaign

Intellectual property laws

• In France : 6 articles of the Intellectual Property Code
• At the European level : 98/44/CE directive & Strasbourg

convention

• At the international level : T.R.I.P.S. agreement

Many laws… Lots of interpretations… Depending on the country… Applicable to all technical fields…

Patent on living

• rganisms

« On a scale of 1 to 5, are you against or for patent on living organisms ? » In total we had 584 replies from all around the world.

5 10 15 20 25 30 35 1 2 3 4 5 % Against For

 We used 4 biobricks from previous teams 2 laccases, T7 promoter and His-tag  We sent 24 biobricks  We created a software « Linker Generator 1.0 »  We proved that the laccase can oxidize the cytochrome C  We produced laccase and cytochrome C

Project achievements

Prototype 2.0 Prototype 1.0

Interlab study

• 6 constructions
• 4 machines

(3 in absolute units, 1 homemade)

Plate reader Fluorimeter Homemade fluorimeter Confocal microscopy

Acknowlegements

HEIDELBERG (Germany) TU DELFT (Netherlands) UAM_Poznan (Poland) SDU-DENMARK (Denmark) UFMG_BRAZIL (Brazil) WESTMINSTER (England)

AMOY (China) BGU ISRAEL (Israel) BIRBECK (UK) GRONINGEN (Netherlands) KU_LEUVEN (Belgium) METU TURKEY (Turkey) NAIT EDMONTON (Canada) OUC CHINA (China)

5 to 10 completed surveys 10 to 30 completed surveys More than 30 completed surveys

SDU-DENMARK (Denmark) UFMG_BRAZIL (Brazil) WESTMINSTER (England)

Acknowlegements

Thank you ! 

• Supervisors
• Advisors
• All the laboratories which helped us
• Generous donors
• iGEM community
• Bordeaux team for the local meetup
• Everyone else who supported us

Thank you ! 

Questions ?

Come to see us ! Hall C, poster 77