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
Phenol
Cadmium
Nitrite
Introduction
PCB
Cadmium
Lead
Spongia officinalis
Phenol
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
Spongia officinalis
Pseudovibrio
denitrificans
Is modifying my
microbiome any
different than
modifying my DNA ?
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
Are you going to
kludge me ?
Unexpected results
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
Resistant
Antibiotic
Effect
Ampicilin
resistant
Tetracyclin
resistant
Chloramphenicol
sensible
Kanamycin
sensible
AMP (200ug.mL-1)
A B
D C
CAM (33ug.mL-1)
A: P.denitrificans
B: Double selection
C: E.coli
D: Transformed E.coli
Sensible
Growth on MB after 72 hours at 30°C with different
antibiotics
Biology
According to the Genome Assembly :
Chemical
Electroporation +
Replicating plasmid
Electroporation + Transposon
EcoKI
Biology
• Tn10 = transposase
• IS10 = repeat inverse
• RFC10 compatible
BBa_K1413044 plasmid map
Biology
MW
1
2
3
4
800
PCR products of kanamycin resistance gene
1, 2, 3 and 4 : clones of P. denitrificans transformed
NT : No transformed P. denitrificans
NT
Biology
Cadmium
Lead
No working BioBricks
RNA Sequencing
Nitrite
PCB
Existing BioBricks
Phenol
(BBa_K1031211)
(BBa_K1031222)
Biology
SfGFP
GFP
ATP
+
BBa K1413001
with GFP’s RBS BBa_B0032
DmpR
DmpR
+
BBa K1413002
with mutated GFP’s RBS
According to Shine Dalgarno motif
Biology
Fluorescence INDUCTION RATIO
vs Bba_K1413002
16000
14000
12000
Induction Ratio
Fluorescence intensity per cell
Fluorescence INTENSITY PER CELL
vs BBa_K1413002
10000
8000
6000
4000
2000
0
1h
6h
11h
1h
6h
11h
BBa_k1413002 (mutated GFP’s RBS):
Better strength
50
45
40
35
30
25
20
15
10
5
0
1h
6h
11h
1h
6h
Better sensitivity
11h
Phenol Model
Phenol
DmpR
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
BioBricks
Submission
BBa_K1413001
Received, Accepted
BBa_K1413002
Received, Accepted
BBa_K1413021
Received, Accepted
BBa_K1413041
Received, Accepted
BBa_K1413043
Received, Accepted
BBa_K1413044
Received, Accepted
• Kludge
• Virtual sponge model
• 6 BioBricks submitted
• 2 Improvements (
• 6 news
)
• Compounds :
• PCB and phenol biosensors
• Phenol Model
• Safety
• Interlab study: 8 promoters characterized
Medal
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
• 1 philosopher
• 2 supervisors