Matthieu Da Costa, William Digan, Cécile Jacry

Matthieu Da Costa, William Digan, Cécile Jacry

Introduction This sphere represents all the water on Earth Its diameter is about 860 miles Wide range of toxic compounds

Introduction PCB Lead Cadmium Phenol Nitrite

Introduction PCB Lead Cadmium Phenol Spongia officinalis Nitrite

Introduction Combine the sponge filtration capacity and its microbiome to detect toxic compounds Filtrates 20 000 L of water/day/kg Epibiosis Are you allowed to modify my DNA? Spongia officinalis Pseudovibrio denitrificans

Introduction Pseudovibrio is the main genus present in sponge microbiome Pseudovibrio denitrificans naturally has a denitrifying metabolism Filtrates 20 000 L of water/day/kg Epibiosis Oh ok! Not me! Spongia officinalis Pseudovibrio denitrificans

Policy and practices Should our sponge be considered as a GMO? GMO GMMO Is modifying my microbiome any different than modifying my DNA? Spongia officinalis Pseudovibrio denitrificans

Policy and practices Epibiosis: a biological containment? Lack of competitivity Strict Epibiosis Physical containment Is Much it contagious better!?

Policy and practices Klumsy Lame Ugly Dumb but Good Enough Engineering biology Unexpected results Are you going to kludge me?

Policy and practices Our goal : Engineering approach Are you going to kludge me? Genome assembly of P.denitrificans Transcriptomic analysis Sponge physiology model

Virtual Sponge Concentration of compound in contact with the sponge microbiome Oscula (out) Ostia (in) Main problems : Complexity of sponge shape Variability of sponge characteristics (number of ostia, oscula) Bacteria location

Virtual Sponge Model 1 : fluxes Model 2: 2D Diffusion Geometry Compound accumulation caused by geometry Result

Genome Assembly 2004 Shieh WY 2006 2008 Enticknap JJ Muscholl-Silberhorn A 2007 Sertan-de Guzman AA 2010 Santos OC igem Evry team 2014 Timeline of articles mentioning Pseudovibrio denitrificans Not very well documented No transformation protocol No genome assembly Genome sequencing Genome assembly First transformation

Genome Assembly Annotation transfer Predicted pathway Nitrate/Nitrite reduction Cadmium resistance Phenol degradation Exogen DNA digestion Antibiotic resistance (Amp&Tet)

Genome Assembly A B C AMP (200ug.mL-1) A B D C Resistant Antibiotic Effect Ampicilin resistant Tetracyclin resistant Chloramphenicol sensible Kanamycin sensible Sensible CAM (33ug.mL-1) A: P.denitrificans B: Double selection C: E.coli D: Transformed E.coli Growth on MB after 72 hours at 30 C with different antibiotics

Biology Chemical Electroporation + Replicating plasmid According to the Genome Assembly : Electroporation + Transposon EcoKI

Biology Tn10 = transposase IS10 = repeat inverse RFC10 compatible BBa_K1413044 plasmid map

Biology MW 1 2 3 4 NT 800 PCR products of kanamycin resistance gene 1, 2, 3 and 4 : clones of P. denitrificans transformed NT : No transformed P. denitrificans

Biology Cadmium Lead Nitrite No working BioBricks RNA Sequencing PCB Phenol Existing BioBricks (BBa_K1031211) (BBa_K1031222)

Biology SfGFP GFP ATP + DmpR DmpR + BBa K1413001 with GFP s RBS BBa_B0032 BBa K1413002 with mutated GFP s RBS According to Shine Dalgarno motif

Induction Ratio Biology 14000 12000 10000 8000 6000 4000 2000 0 Fluorescence intensity per cell16000 Fluorescence INTENSITY PER CELL vs BBa_K1413002 1h 6h 11h 1h 6h 11h 50 45 40 35 30 25 20 15 10 5 0 Fluorescence INDUCTION RATIO vs Bba_K1413002 1h 6h 11h 1h 6h 11h BBa_k1413002 (mutated GFP s RBS): Better strength Better sensitivity

Phenol Model DmpR Phenol ATP DmpR dimer No phenol degradation Phenol degradation

Interlab Study Corrected GFP fluorescence intensity according to OD 600 nm Corrected GFP Fluorescence at OD 600 nm = 0.45 Characterized required promoters (I020260, J23101 and K823012 ) 8 characterized out of 18 promoters tested in the Anderson Library

Achievements Pseudovibrio denitrificans : First marine bacteria in igem First transformation Genome sequencing/assembly Kludge Virtual sponge model BioBricks Submission Medal BBa_K1413001 Received, Accepted BBa_K1413002 Received, Accepted 6 BioBricks submitted 2 Improvements ( ) 6 news Compounds : PCB and phenol biosensors Phenol Model BBa_K1413021 BBa_K1413041 BBa_K1413043 BBa_K1413044 Received, Accepted Received, Accepted Received, Accepted Received, Accepted Safety Interlab study: 8 promoters characterized

Acknowledgement Lab facilities: Institute of Systems & Synthetic Biology (issb, Evry) DNA /RNAs sequencing facilities: CEA (Genoscope, Evry) Sponge microbiome advices: MDCEM team of the French National Museum of Natural History (MNHN, Paris) Conjugation plasmid: Institute of Molecular Enzyme Technology, Group of Bacterial Photobiotechnology (Heinrich-Heine-Universität, Düsseldorf) Sponge expertise: Mediterannean Institute of Biodiversity and Ecology (IMBE, Marseille)

Acknowledgement 4 advisors 9 biologists 3 bioinformaticians 2 supervisors 1 philosopher