Outils pour la Biologie de synthèse :Micro et Milli fluidique!

Outils pour la Biologie de synthèse :Micro et Milli fluidique !

Jérôme  Bibe+e

Laboratoire  Colloïdes  et  Matériaux  Divisés

ESPCI  ParisTech

Must be suitable for micro organism growth: example of yeast growth.!

t = 0 t = 17 h

1 mm

Croissance d’amas cellulaires!

collaboration avec l’Institut Curie (ANR CAPCELL)

Cellules ct26 (cellules cancéreuses de souris)

!   Study  over  numerous  cells:  sta2s2cal  power  

RDT-­‐  Confiden2al  

10X  

4X  

2X  

!  

Glucose, salts!

!  

A!

low solubility! high solubility!

‣ 

Requirements

At the time scale of the experiment (A few hours)

1) Nutrients (salts, carbohydrates) must not permeate

2)Products of the bioactivity (CO2, Ethanol, etc) must permeate almost instantaneously, or get trapped or degraded into cells (ATP) 3)Relaxation of osmotic missmatch must be governed by water permeation only

HOW TO MAKE SUCH SYSTEM? And

make it extremely sensitive and quantitative

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CELL COMPARTEMENTALIZATION

λ=1/10 λ=1/5 λ=1/2

Confined  2d  Droplet  Array  

100  µm  

x  

y   x   y  

100  µm   4  cm  

40  µm  

10  

Limiting factor Exponential growth

Constant yield

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~600 colonies

time (min) V (pL)

: substrat quantity

time (min)

Y=0.15g(biomasse)/gglucose ; τ =120 min.

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V/V0

time (min)

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For time (min)

V (pL)

time (min)

Slope is conserved Volume follows substrat consumption

: substrat quantity

68

V/V0

Time (min)

~ single cell glucose consumption

Single cell bloked in G1 phase

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95

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Strain Growth Ethanol

Turbo yeast 48 +++ ++

CEN PK ++ +/-

TMB 3001 + +/-

C5 + -

Y55 +/- ++

CLIB 413 Agregats -

YJM 454 - ++

CLIB 192 - +++

(Batch experiment, Raphaël Calbrix)

102

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Strain Growth Ethanol

Turbo yeast 48 +++ ++

CEN PK ++ +/-

TMB 3001 + +/-

C5 + -

Y55 +/- ++

CLIB 413 Agregats -

YJM 454 - ++

CLIB 192 - +++

(Batch experiment, Raphaël Calbrix)

time (min) V*

103

‣ 

Strain Growth Ethanol

Turbo yeast 48 +++ ++

CEN PK ++ +/-

TMB 3001 + +/-

C5 + -

Y55 +/- ++

CLIB 413 Agregats -

YJM 454 - ++

CLIB 192 - +++

(Batch experiment, Raphaël Calbrix)

time (min) V*

104

‣ 

Strain Growth Ethanol

Turbo yeast 48 +++ ++

CEN PK ++ +/-

TMB 3001 + +/-

C5 + -

Y55 +/- ++

CLIB 413 Agregats -

YJM 454 - ++

CLIB 192 - +++

(Batch experiment, Raphaël Calbrix)

time (min) V*

105

‣ 

Strain Growth Ethanol

Turbo yeast 48 +++ ++

CEN PK ++ +/-

TMB 3001 + +/-

C5 + -

Y55 +/- ++

CLIB 413 Agregats -

YJM 454 - ++

CLIB 192 - +++

(Batch experiment, Raphaël Calbrix)

time (min) V*

106

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glucose

(maize, beet, sugar canne, starch...)

fermentation

ETHANOL 1^st generation bioethanol:

2^nd generation bioethanol: cellulose & lignocellulose

(forests, wastes, miscanthus...)

saccharification

sugar (C6+C5)

fermentation

ETHANOL enzymes

NEW STRAINS

116

‣ 

117

V*

time (min)

Strain Growth Ethanol

Turbo yeast 48 +++ ++

CEN PK ++ +/-

TMB 3001 + +/-

C5 + -

Y55 +/- ++

CLIB 413 Agregats -

YJM 454 - ++

CLIB 192 - +++

25g/L glucose - 25g/L xylose

LCMD-ESPCI

Laurent Boitard Denis Cottinet Nicolas Bremond

Enric Santanach-Carreras Leslie Rolland (PhD)

Hugo Doméjean (PhD) Institut Curie-UMR 168

Pierre Nassoy

Christophe Lamaze Kévin Alessandri(PhD) Bibhu-Ranji Sarangi

THANKS

Co-extrusion dans l’air!

Fragmentation

Cœur hydrophobe : liquides non mouillants

Cœur aqueux : liquides miscibles

Gélification

Goutte d’eau!

Goutte d’eau + SDS!

La précipitation du SDS à l’interface inhibe

le mélange avec le bain de gélification.

Cœur aqueux , alginate + tensioactif (SDS)!

membrane fine, h ~ 50 µ m

Growth Mechanics, HTS against

therapeutic candidates

Capsules sub-millimétriques!

Forçage de la déstabilisation capillaire d’un jet composé de 2 liquides

d

= 200 µ m f = 1.5 kHz

Capsules:

Supplementary Figure 4

Growth of spheroids inside alginate capsules and after capsules bursting.

(a) Representative plots showing the time evolution of the spheroid’s radius RMCS normalized to the initial inner capsule radius R0 for different CT26 spheroids grown in thin capsules. Time t=0 is taken at confluency. The steep increase of RMCS in the later stages corresponds to capsule bursting. The spheroid grows freely at a rate similar to that observed in the very early stages following encapsulation. (b) Typical sequence of phase contrast images showing the different stages of MCS growth, before confluency, after confluency, and after capsule bursting. Scale bar, 100 µm.

Croissance d’amas cellulaires!

collaboration avec l’Institut Curie (ANR CAPCELL)

-  Mesure de la pression exercée par la croissance d’un agrégat

-  Trafic intra-cellulaire au sein d’un tissu confiné