team : aix-marseille project2015.igem.org/files/poster/aix-marseille.pdf · 2015. 10. 21. · team...
TRANSCRIPT
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.