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

Team Aix-Marseille Team Aix-Marseille 2015 2015 Chew Fight

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

Did you know ? 374 billion 100,000 tons of chewing gum of chewing gum are consumed each year sticks are made every year

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 CH 2 CH 2 C CH Sugar CH 3 20% n Polyisoprene Gum base Syrup 60% 20% CH2 CH CH2 CH CH CH2 Polystyrene butadiene 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

Which enzymes ? Latex clearing protein • Specific metal oxidase

Which enzymes ? 𝟐 𝟐 H + + 𝟑 H 2 O Ruthenium 𝟓 O 2 Laccase Cu II Cu I • General metal oxidase Schneider, Ludovic, et al. "Visible‐Light‐ Driven Oxidation of Organic Substrates with Dioxygen Mediated by a [Ru (bpy) 3] 2+/Laccase System." ChemSusChem (2015)

Our solution 𝟐 𝟐 H + + 𝟑 H 2 O 𝟓 O 2 Laccase Cytochrome C Cu II Cu I Fe II Fe III • Oxydase • Hydroxylase

Proposition 𝟐 H + + 𝟓 O 2 𝟐 𝟑 H 2 O Reduced Laccase Cu I Oxidised laccase Cu II Very oxidised Cyt C Fe IV hv Oxidised cyt C * R Fe III * . + O O 2 R R Reduced laccase 𝟐 H + + Oxidised Cyt C 𝟓 O 2 Cu I Fe III Reduced Cyt C 𝟐 𝟑 H 2 O Oxydised Laccase Fe II Cu II

Proof of concept Abs (550 nm) Laccase can oxidize Cytochrome C

Our constructions G. polyisoprenivorans pT7 - RBS LCP His Streptomyces sp K30 E. coli pT7 - RBS Laccase His T. thermophilus B. Subtilis P. denitrificans pT7 - RBS Cytochrome C His S. oneidensis Synechosystis PCC 6803 pT7 - RBS Laccase Linker Cytochrome C His pT7 = T7 promoter RBS = Ribosome binding site LCP = Latex clearing protein

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

Laccase results P T7 - RBS Laccase His E. coli Ladder IPTG kDa kDa 3 Purification Western blot revealed SDS-PAGE by antibody against His- Comassie brillant blue tag Chassis BL21 (DE3)

Laccase activity Laccase from ISM2 ABTS without laccase [O2] (%) Our laccase with ABTS Our laccase incubated 20 min with 100 mM copper Time (s)

Cytochrome C results P T7 - RBS Cytochrome C His S. oneidensis 1- Ladder 2- Cytoplasm 3- Solubilized membranes Western blot revealed by antibody against His-tag

Next step …

Human practices

Celibrities A.L W.S W.W W.W Jr - 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

Next celibrities ?

Applications 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

Our product  Eco-responsible  Saves Water  Saves Energy  Saves Money  No chemical products  Major issue : solution based on GMOs

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

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 organisms « 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. % 35 30 25 20 15 10 5 0 1 2 3 4 5 Against For

Project achievements  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

Prototype 2.0 Prototype 1.0

Interlab study Fluorimeter Plate reader  6 constructions  4 machines (3 in absolute units, 1 homemade) Confocal Homemade microscopy fluorimeter

More than 30 completed surveys Acknowlegements AMOY (China) BGU ISRAEL (Israel) 10 to 30 completed BIRBECK (UK) GRONINGEN (Netherlands) surveys KU_LEUVEN (Belgium) METU TURKEY (Turkey) NAIT EDMONTON (Canada) 5 to 10 completed OUC CHINA (China) surveys SDU-DENMARK (Denmark) UFMG_BRAZIL (Brazil) WESTMINSTER (England) HEIDELBERG (Germany) TU DELFT (Netherlands) UAM_Poznan (Poland)

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

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