Matthieu Da Costa, William Digan, Ccile Jacry Introduction This - - PowerPoint PPT Presentation

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Matthieu Da Costa, William Digan, Ccile Jacry Introduction This - - PowerPoint PPT Presentation

Mar 12, 2024 469 likes •736 views

Matthieu Da Costa, William Digan, Ccile Jacry Introduction This sphere represents all the water on Earth Its diameter is about 860 miles Wide range of toxic compounds Introduction PCB Cadmium Lead Nitrite Phenol Introduction PCB

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Matthieu Da Costa, William Digan, Cécile Jacry

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

Introduction

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Cadmium Phenol Lead Nitrite PCB

Introduction

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Cadmium Phenol Lead Nitrite PCB Spongia officinalis

Introduction

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Spongia officinalis Filtrates 20 000 L of water/day/kg Pseudovibrio denitrificans Combine the sponge filtration capacity and its microbiome to detect toxic compounds Epibiosis

Are you allowed to modify my DNA ?

Introduction

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Spongia officinalis Filtrates 20 000 L of water/day/kg Pseudovibrio denitrificans

 Pseudovibrio is the main genus present in sponge microbiome  Pseudovibrio denitrificans naturally has a denitrifying metabolism

Epibiosis

Introduction

Oh ok ! Not me !

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Spongia officinalis Pseudovibrio denitrificans Should our sponge be considered as a GMO ?

Is modifying my microbiome any different than modifying my DNA ?

≠ GMO GMMO

Policy and practices

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Epibiosis: a biological containment?

  • Lack of competitivity
  • Strict Epibiosis
  • Physical containment

Is it contagious ? Much better !

Policy and practices

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Engineering biology Unexpected results Klumsy Lame Ugly Dumb but Good Enough

Are you going to kludge me ?

Policy and practices

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  • Genome assembly of P.denitrificans
  • Transcriptomic analysis
  • Sponge physiology model

Our goal : Engineering approach

Policy and practices

Are you going to kludge me ?

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Main problems :

  • Complexity of sponge shape
  • Variability of sponge characteristics

(number of ostia, oscula)

  • Bacteria location

Concentration of compound in contact with the sponge microbiome Ostia (in) Oscula (out)

Virtual Sponge

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Result Geometry

Compound accumulation caused by geometry

Model 1 : fluxes Model 2: 2D Diffusion

Virtual Sponge

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Timeline of articles mentioning Pseudovibrio denitrificans

2004

Shieh WY

2006

Enticknap JJ

2007

Sertan-de Guzman AA

2008

Muscholl-Silberhorn A

2010

Santos OC

iGEM Evry team 2014

  • Not very well documented
  • No transformation protocol
  • No genome assembly

 Genome sequencing  Genome assembly  First transformation

Genome Assembly

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  • Nitrate/Nitrite reduction
  • Cadmium resistance
  • Phenol degradation
  • Exogen DNA digestion
  • Antibiotic resistance (Amp&Tet)

Predicted pathway Annotation transfer

Genome Assembly

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Growth on MB after 72 hours at 30°C with different antibiotics

A: P.denitrificans B: Double selection C: E.coli D: Transformed E.coli

Genome Assembly

CAM (33ug.mL-1)

A B C D A B C

AMP (200ug.mL-1)

Sensible Resistant

Antibiotic Effect Ampicilin resistant Tetracyclin resistant Chloramphenicol sensible Kanamycin sensible

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

Biology

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BBa_K1413044 plasmid map

  • Tn10 = transposase
  • IS10 = repeat inverse
  • RFC10 compatible

Biology

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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

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Cadmium Lead Nitrite PCB Phenol No working BioBricks RNA Sequencing Existing BioBricks

(BBa_K1031211) (BBa_K1031222)

Biology

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DmpR +

DmpR

+

ATP

SfGFP

BBa K1413002 with mutated GFP’s RBS According to Shine Dalgarno motif BBa K1413001 with GFP’s RBS BBa_B0032 GFP

Biology

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Biology

5 10 15 20 25 30 35 40 45 50 Induction Ratio

Fluorescence INDUCTION RATIO vs Bba_K1413002

1h 6h 11h 1h 6h 11h

Better sensitivity BBa_k1413002 (mutated GFP’s RBS): Better strength

2000 4000 6000 8000 10000 12000 14000 16000 Fluorescence intensity per cell

Fluorescence INTENSITY PER CELL vs BBa_K1413002

1h 6h 11h 1h 6h 11h

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Phenol ATP

DmpR

DmpR dimer

Phenol Model

Phenol degradation No phenol degradation

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  • Characterized required promoters (I020260, J23101 and K823012 )
  • 8 characterized out of 18 promoters tested in the Anderson Library

Corrected GFP Fluorescence at OD 600 nm = 0.45

Interlab Study

Corrected GFP fluorescence intensity according to OD 600 nm

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BioBricks Submission Medal BBa_K1413001 Received, Accepted BBa_K1413002 Received, Accepted BBa_K1413021 Received, Accepted BBa_K1413041 Received, Accepted BBa_K1413043 Received, Accepted BBa_K1413044 Received, Accepted

  • Pseudovibrio denitrificans :
  • First marine bacteria in iGEM
  • First transformation
  • Genome sequencing/assembly
  • Kludge
  • Virtual sponge model
  • 6 BioBricks submitted
  • 2 Improvements ( )
  • 6 news
  • Compounds :
  • PCB and phenol biosensors
  • Phenol Model
  • Safety
  • Interlab study: 8 promoters characterized

Achievements

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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)

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  • 9 biologists
  • 3 bioinformaticians
  • 1 philosopher
  • 4 advisors
  • 2 supervisors

Acknowledgement