ProjectTeam : Aix-Marseille

CamilleProject manager

Marion Project manager

EllaProject member

Axel Project manager

YoannProject manager

SébastienEnzymologist

MyriamProject member

DanielProject member

YassineProject member

LaureBeyond the bench

WilsonSoftware and

modelling

Sponsors

Introduction

Problems linked to chewing gum

Do not worry, you are not the only ones concerned !

1- Chewing gum composition

2- Our strategy

3- How to degrade chewing gum ?

4- Our constructions

Gum base

Sugar20%

Syrup20%

24%

18%

19%7%

8%

5%19%

resins

syntheticresinswax

solid polymers

liquidpolymersemulsifyingagent

It’s all about oxidations and reductions reactions between laccases and cytochrome C….

Human practices

Interlab study

Results

Cytochrome C Laccase

DNA constructions were verified by PCR, digestion and sequencing,Proteins were produced in aerobic conditions and purify by affinity chromatography with theHis-tag.

18

28

38

49

kDa

14

6

1 2 3 4 5

Enzymatic activity

Question: Can the laccase oxidize the cytochrome C ?

Laccase can oxidize cytochrome c

Software & ModellingModelling allowed us to design linkers between all our selected laccases and cytochromes C.

Linker between Laccase E. coli and CytC S. oneidensis

We created a software toolwhich generates linkers in orderto calculate the minimal distancebetween two proteins. Let’s tryit!

Confocal microscope Magicbox (home made filter based fluorimeter)

We choose to use 4 different methods to study the strength of 5 different promoters

Fluorometer (SPEX fluorlog)TECAN microplate reader

TECAN Fluorometer MagicboxConfocal

Microscopy

J23100 +++++ +++++ +++++ +++++

J23101 ++++ ++++ +++ +++++

J23118 ++++ +++ +++++ +++++

J23106 ++ ++ + +

J23117 + + + +

P T7 - - - -

Our results : J23100 is the strongest combination compared to the negative control PT7.

All the machines except the confocal microscope were calibrated using a standard solution of the dye Atto488. This allows us to compare the measurements on the different instruments.

Acknowledgement :

James Sturgis, Laboratory director and professor ; Gaël Chambonnier, PhD candidate ; Romain Clément, PhD candidate ; Laureen Logger, PhD candidate ; Valérie Prima, engineer ; Victoria Schmidt, PhD candidate ; Bastien Serrano, engineer ; Olivier Uderso, medium preparatorAnnick Brun, maintenance ; Isabelle Bringer, maintenance ; Laetitia Houot, associate professor ; Jean-Pierre Duneau, associate professor ; Anne Lomascolo, associate professor ; Pedro Coutinho, associate professor ; Catherine Heilmann, laboratory maintenance ; Marius Reglierand Thierry Tron from ism2 (institut des sciences moléculaires de Marseille) Edouard Tourdot, student of Team Bordeaux

Cytochrome C production

Laccase production

Laccase purification

The test didn’t show theenzymatic activity of the purifiedlaccase

We designed linker between laccase and cytochrome C to optimize electron transfer

styrene butadienepolyisoprene

SimonBeyond the bench

Additionnal constructions to improve export (CCHL) and folding (HemA)

1 2 3 4 5

1 trillionchewing gums are stuck everyday on the street

100,000 tons of chewing gum are consumed each year

Did you know ?

What did you expect ?When you eat chewing gums you actually eat

plastic…

Toxic for wild life and for the environmentA large quantity of water,

energy and time are wastedto clean it

It takes 5 years to be degraded

Cities spend millions of dollars per year to clean it

Second pollutant after cigarette butts

Chewing gum is composed of two parts :- Sugar and syrup (Hydrophilic part) degraded by saliva- Gum base (Hydrophobic part) OUR project !!

Gum base is composed of many polymers :

Time (sec)

[O2] (

Light-driven polymer degradation by Laccase-Ruthenium system

Synthetic biology

Laccase Ru+ +

CytC

CytC

Laccase

1- The MarketThanks to various testimonies (notablyONET, first french cleaning company) weknow that an eco-responsible product willfind a place in the market.

Thanks to Groningen teamto share with us their urban pollution.

2- Product’s image

3- What about patents ?

The intellectual property code contains a huge number of laws differentlyinterprated by the population. To commercialize our product, we shouldpatent it and go beyond national laws transcription problems.

Against

The opinions are globally divided about GMOs’ utilization. InEurope GMO utilization soundsnot as good as in USA. So, it isdifficult to introduce our productwithout a strong communicationcampaign.

Laccase Lypoxygenase

Pro

Against or pro GMO’s utilization ?

Cytochrome C replaces Rutheniumrole. To improve polymer

degradation, Lipoxygenase is added.