ac citrico.pdf

8/17/2019 ac citrico.pdf

1/64

La Revolución dela Biotecnología

Prof. Juan A. Asenjo


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• Edward Jenner (1749 –1823): “cowpox” – smallpox – Vacuna viruela

• 1850 Luis Pasteur:

Microorganismos: fermentación no es espontánea

• 1928: Alejandro Flemming : Penicilina

• 1939: Florey, Chain purificación de penicilina y producción masiva

USA-Pfizer Producción de ácido cítrico

levadurasfermentación

Esterilización (descubrió los microorganismos)(Enzimas)

• 1945: Premio Nobel: Flemming, Florey, Chain

azúcar

levadura

CO2 + H 2O

alcohol


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Obtención de Plasmidios

2.- Sacar plasmidio desde bacteria

1.- Se cuenta con bacterias que contienen plasmidios

Cromosoma

Plasmidio

Bacteria

Plasmidios

Poración

Producción & Purificación de Proteínas


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Principales pasos en la Clonación deun Segmento de DNA Foráneo


1.- Obtención del DNA foráneo

2.- Corte con Enzimas de restricción del plasmidio

Extremos

cohesivos

Extremos

cohesivos

Plasmidio

Corte

Plasmidio Cortado

(Enzimas de

Restricción)

Extremos

cohesivos


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• 60’s - 70’s Ingeniería Genética

• 80’s INSULINA: Ingeniería genética de E.coli y S.cerevisiae

Insulina comercial recombinante

• Hoy: Eli-Lilly

– Novo-Nordisk

• 90’s: tpA

• Vacunas: Contra hepatítis B (Merck, Chiron)

Sida

• 1990 Sally y Dolly• Enzimas criofílicas

• Terapia celular y génica


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Nueva Biología Molecular

Proteínas “Clonadas”

• Ingeniería Genética

– Enzimas de Restricción

– Plasmidios



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Principales pasos en la Clonación deun Segmento de DNA Foráneo


1.- Obtención del DNA foráneo

2.- Corte con Enzimas de restricción del plasmidio

Extremos

cohesivos

Extremos

cohesivos

Plasmidio

Corte

Plasmidio Cortado

(Enzimas de

Restricción)

Extremos

cohesivos


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4.b.- Introducción del plasmidio Recombinante en célula anfitriona

⇒

Permeasa

4.- Transformación

4a.- Permeabilización de la célula mediante permeasa



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Biotecnología• Nueva Biología Molecular

• Proteínas “Clonadas”

• Ingeniería de Proteínas

• Ingeniería Metabólica (Systems Biology)

• Genómica Funcional

• Nuevos Productos Terapéuticos

• Nuevas Vacunas• Nuevas Enzimas Industriales

• Cultivo de Tejidos, Terapia Génica


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We haven’t the money, so we’ve got to

think

Ernest Lord Rutherford, 1871 - 1937


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Systems Biology


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Ogni parte ha

inclinazione di

ricongiungersi al

suo tuttoper fuggire dalla

sua imperfezione

Leonardo da Vinci(Cod.Atl, fol 59 recto)


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The part always

has a tendency

to reunite with

its whole in order to escape from

its imperfection

Leonardo da Vinci(Cod.Atl, fol 59 recto)


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Estructura de las Proteínas

• Estructura Primaria: secuencia lineal de aa

• Estructura Secundaria: algunos aa interactuan

• Estructura Terciaria: cadenas de aa interligadas

• Estructura Nativa: proteína se encuentra activa

• Proteína denaturada:

– No tiene actividad – No posee puentes disúlfuro



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ProteínasCuatro niveles de

estructura:desde 1 dimensión

a 3 dimensiones

Desde análisis

estructural

a análisis funcional


18/64

Ingeniería de Proteinas: Predicción Estructural


19/64

Ingeniería de Proteínas


20/64


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Proteasa criofílica antártica


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• Proteasas activas a baja temperatura(Criofílicas, Psicrofílicas)

• para detergentes

• para industria de alimentos

• Para aplicaciones médicas


23/64


• Estudios de Relación Estructura-Función

• Mutagénesis Sitio-Dirigida

• Mutagénesis al Azar


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Mutagénesis al azar (random)

Evolución dirigida

Mutagénesis de saturación

“Gene shuffling”(“barajar” genes)


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Proteasa criofílica antártica


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Is there a Rational Method to

Purify Proteins?

from Expert Systems toProteomics

J.A. Asenjo

University of Chile


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The Combinatorial Characteristic of Choosing the

Sequence of Operations for Protein Purification

Third

Stage

C1

C2

C3

C5

C6

n th

Stage

n1

n2

n3

n5

n6

Second

Stage

B1

B2

B3

B4

B5

B6

First

Stage

A1

A2

A3

A4

A6

1) Ion Exchange

Chromatography

3) Affinity

Chromatography

4) Aqueous Two-

Phase Separation

5) Gel Filtration

2) Hydrophobic

Interaction

Chromatography

6) HPLC


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FactsRules

Knowledge base Workingmemory

Knowledgeacquisition

subsystem

ControlInference

Inference engine

Userinterface

Explanationsubsystem

Expert orKnowledge

engineer

User

The architecture of a knowledge based expert system. (taken from

Asenjo, Herrera and Byrne, 1989)


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Determination of the Resolution Between Two Peaks

V2-V1

½(W1+W2)

RS =

SC α RS

η =

DF DF

SC α RS

V1

V2

W1 W2

A b s o r b a n c e

Time


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The model of database components for main protein contaminants in one of the

production streams to be used in the selection of optimal separation operation

CHARGE

PROTEINS

PRODUCT

CONTAMINANT 1

CONTAMINANT 2

CONTAMINANT 3

CONTAMINANT 4

CONTAMINANT N

pH 4.0 pH 4.5 . . . . pH 9.5 pH 10.0

PROPERTYCONCENTRATION

MOLECULAR

WEIGHTISOELECTRIC

POINT

HYDROPHO-

BICITY

CONTAMINANT 5.

.

..

.

.


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Concentration, molecular weight, hydrophobicity and charge at different pHs, for the main

proteins (“contaminants” of the product) in Escherichia coli. Data from Woolston (1994)

Contaminant

Cont_1

Cont_2

Cont_3

Cont_4

Cont_5

Cont_6

Cont_7

Cont_8

Cont_9

Cont_10

Cont_11

Cont_12

Cont_13

pH 7

q G

-2.15

-3.50

-0.85

-1.73

-3.07

-3.05

-1.00

-3.32

-0.21

-0.53

0.05

0.50

1.50

g/litre

weight

11.29

7.06

4.63

5.58

4.83

2.48

7.70

6.80

7.53

6.05

3.89

1.48

0.83

pI 1

4.67

4.72

4.85

4.92

5.01

5.16

5.29

5.57

5.65

6.02

7.57

8.29

8.83

Da

Mol wt 2

18,370

85,570

53,660

120,000

203,000

69,380

48,320

93,380

69,380

114,450

198,000

30,400

94,670

*

hydroph 3

0.71

0.48

0.76

1.50

0.36

0.36

0.48

0.93

0.63

0.06

pH 4

q A

1.94

2.35

1.83

3.29

4.08

5.22

3.96

10.90

1.09

10.40

0.33

5.17

11.70

pH 4,5

q B

0.25

0.29

0.67

1.38

1.83

3.17

3.16

5.81

0.55

5.94

0.03

4.22

7.94

pH 5

q C

-0.80

-1.17

0.04

-0.03

0.04

1.02

1.12

2.78

0.26

3.15

0.05

3.20

5.39

pH 5,5

q D

-1.41

-2.17

-0.30

-0.69

-1.17

-0.72

-0.58

0.77

0.10

1.51

0.05

2.25

3.73

pH 6

q E

-1.76

-2.83

-0.49

-1.07

-1.92

-1.90

-1.36

-0.81

-0.03

0.56

0.05

1.46

2.66

pH 6,5

q F

-1.97

-3.24

-0.65

-1.34

-2.46

-2.60

-1.34

-2.18

-0.12

-0.05

0.05

0.87

1.97

pH 8,5

q J

-2.67

-3.64

-1.50

-2.75

-5.65

-4.24

-2.84

-4.31

-0.32

-1.72

-1.57

0.08

0.51

pH 7,5

q H

-2.33

-3.63

-1.90

-2.30

-3.90

-3.46

-0.95

-4.12

-0.28

-0.99

-0.69

0.30

1.13

pH 8

q I

-2.45

-3.68

-1.34

-2.85

-4.98

-3.90

-1.59

-4.45

-0.32

-1.43

-0.97

0.20

0.80

Charge4 (Coulomb per molecule x 1E25)

* Hydrophobicity expressed as the concentration (M) of ammonium sulphate at which the protein eluted.

(Higher values represent lower hydrophobicity).1 Measured by isoelectric focusing using homogeneous poolyacrylamide gel in Phast System.2Molecular weight was measured by SDS-PAGE with PhastGel media in Phast System.3Hydrophobicity was measured by hydrophobic interaction chromatography using a phenyl-superose gel in an

FPLC and a gradient elution from 2.0 M to 0.0 M (NH4)2SO4 in 20 mM Tris buffer.4Charge was measured by electrophoretic titration curve analysis with PhastGel IEF 3-9 in a Phast System.


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DFi

DFi

B

C AS

A

B

b

DFi

B

C A S

DFi

C

S

A

B

D

b´

Representation of the peaks of a chromatogram as triangles, showing how the variation in the value of DF

leads to different concentrations of the contaminant protein in the product. The triangle on the left

corresponds to the product protein and the triangle of the right corresponds to the peak of the protein

being separated (contaminant).


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Genomics

Proteomics

Metabolomics

Economics


36/64

Genes

Proteínas

Metabolismo

Sistemas Multicelulares

Organismo


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MetabolómicaIngeniería Metabólica

• Systems Biology: qué viene

después de la Genómica

• Uso de Análisis de Flujos

Metabólicos y Tecnología de

Microarrays de Genes

M t b l i


39/64

MetabolomicsGLUCGLUC

GLUC6PGLUC6P

FRUC6PFRUC6P

3PG3PG

GAPGAP

PIRPIR

PEPPEPACETACETEtOHEtOH

ACAC

RIBU5PRIBU5P

XIL5PXIL5PRIB5PRIB5P

GAPGAPSED7PSED7P

FRUC6PFRUC6P

aaaa

aaaa

aaaa

aaaa

aaaaaaaaE4PE4P

CARBCARB

ATP ADPATP ADP

RNARNA

OO22EE OO22

COCO22 COCO22EE

υ 2

υ 3

υ 5

LIPLIP

AcCoAAcCoAmitmit

AcCoAAcCoAcitcit

FUMFUM AKGAKG

SUCCoASUCCoASUCSUC

MALMAL ISOCITISOCIT

OACOAC

SODSOD

SODSOD

SODSOD

SODSOD

SODSOD

PROTPROTPROTPROT

PROTPROT

PROTPROT

PROTPROT

υ 6

υ 7

υ 9

υ 13

υ 11

υ 10

υ 10

υ 76

υ

77

7 0 - a a O

A C

υ 69

7 1 - a a O AC

υ 17

υ 16

υ 15

υ 14

υ 73-AcCoA

υ

30

υ 70-aaAKG

υ 71-aaAKG

υ 70-aaPIR

υPEP

υPIR

υ 74

υ 31

υ 3PG

υ 28

υ 27

υ 26

υE4P

υ 19 υ20

υ 21

υ 22

υ 23

υ 18 υ1

υ 25

υ

71-aaPIR

υ 70-aa3PG

υ 71-aaPEP

υ 70-aaPEP

υ 71-aa3PG

υ 71-aaE4P

υ70-aaE4P

υ 70-aaR IB5P

υ71-aaRIB5P

υ 72-nuOAC

υ 72-nuRIB5P

υ 72-nu3PG

NHNH44EE NHNH44

υ 78

LIPLIP

7 3 - G A

P

PROTPROTaaaa

RNARNA SODSOD

nunu

υOAC

nunuυRI B5P

aaaa

υAc CoAcit

υ 71-aaAcCoA

υ 70-aaAcCoA

υAK G

RNARNA

nunu

GLICGLIC

AcCoAAcCoAcitcit

υ 24

υ 75

υ 4

υ 8


40/64

dX/dt = S v - bdX/dt = S v - b

in SS: S v = bin SS: S v = b oror S r = 0S r = 0 SScc rrcc + S+ Smm rrmm = 0= 0

Metabolic Flux AnalysisMetabolic Flux AnalysisMetabolic Flux BalanceMetabolic Flux Balance

AA

EE

BB

CC

DD FF

ν

ν

3

ν

2

ν

5

ν

4

S r=0=S r=0=1-0100D

01-010C

001-1-1B

54321 ν ν ν ν ν

5

4

3

2

1

ν

ν

ν

ν

ν

100D

010C

1-1-1B

321 ν ν ν

3

2

1

ν

ν

ν

1-0D

01-C

00B

54 ν ν

5

4

ν

ν

+

SS StoichiometricStoichiometric MatrixMatrix

rr Rate (Flux) vectorRate (Flux) vector

cc CalculatedCalculated

mm MeasuredMeasured

S i PS i P S i PS i P


41/64

0

3

6

9

12

15

0 9 18 27 36 45Time, h

G l u c o s e , g / L

0.0

0.7

1.4

2.1

2.8

3.5

C e l l s ,

E t h a n o l a n d S O D , g / L

Strain P+Strain P+ Strain PStrain P--

0

3

6

9

12

15

0 9 18 27 36 45

Time, h

G l u c o s e , g / L

0.0

0.7

1.4

2.1

2.8

3.5

C e l l s a n d E t h a n o l , g / L

0.0

0.3

0.6

0.9

1.2

1.5

0 9 18 27 36 45

Time, h

T o t a l P r o t e i n a n d C a

r b o h y d r a t e s , g / L

0.00

0.05

0.10

0.15

0.20

0.25

T o t a l R N A , g / L

Strain P+Strain P+ Strain PStrain P--

0.0

0.3

0.6

0.9

1.2

1.5

0 9 18 27 36 45Time, h

T o t a l P r o t e i n a n d C a r b

o h y d r a t e s , g / L

0.00

0.05

0.10

0.15

0.20

0.25

T o t a l R N A ,

g / L


42/64

P+ GLUC

GLUCGLUC

GLUC6PGLUC6P

FRUC6PFRUC6P

3PG3PG

GAPGAP

PIRPIR

PEPPEP

ACETACETEtOHEtOH

RIBU5PRIBU5P


GAPGAPSED7PSED7P

FRUC6PFRUC6P

aaaa

aaaa

aaaa

aaaa

E4PE4P

CARBCARB

3.844

4.169

6.256

RNARNA

GLICGLIC

SODSOD

SODSOD

SODSOD

PROTPROTPROTPROT

6.151

6.122

0.029

0.138

0.208

2.232

0.105

4.130 4.267

0.029

0.234 0.325

0.177

0.148

0.559 4.611

0.017

0.048

0.004

0.025

0.028

0.0040.025

0.006 0.0060.042

0.019

LIPLIP

0 . 0 0 2

PROTPROTaaaa

RNARNASODSOD

nunu

nunu

0.057

0.177

PEPPEP aaaaaaaa SODSODPROTPROT . 0.025

0.0040.025


43/64

P+ GLUC

GLUCGLUC

GLUC6PGLUC6P

FRUC6PFRUC6P

3PG3PG

GAPGAP

PIRPIR

PEPPEP

ACETACETEtOHEtOH

ACAC

RIBU5PRIBU5P


GAPGAPSED7PSED7P

FRUC6PFRUC6P

aaaa

aaaa

aaaa

aaaa

aaaa

aaaa

E4PE4P

CARBCARB

ATP ADPATP ADP

RNARNA

OO22EE OO22

COCO22 COCO

22EE

3.844

4.169

6.256

LIPLIP

AcCoAAcCoAmitmitAcCoAAcCoAcitcit

FUMFUM AKGAKG

SUCCoASUCCoASUCSUC

MALMAL ISOCITISOCIT

OACOAC

RNARNA

GLICGLIC

SODSOD

SODSOD

SODSOD

SODSOD

SODSOD

PROTPROTPROTPROT

PROTPROT

PROTPROT

PROTPROT

6.151

6.122

1.470

8.850

3.564

0.079

8.988

0.025

0.121

0.102

0.166

0.097

0.023

0.069

0.029

0.138

0.208

2.232

0.105

0.137

4.130 4.267

0.029

0.234 0.325

0.177

0.148

0.559 4.611

0.247

0.017

0.048

0.004

0.025

0.028

0.0040.025

0.006 0.006

0.022

0.042

0.019

NHNH44EE NHNH44

0.724

LIPLIP

0 . 0 0 2

PROTPROTaaaa

RNARNASODSOD

nunu

nunu

0.174

nunu

0.057

aaaa0.063

0.014

0.046

1.470

1.470

1.470

1.345

1.3491.349

1.397

1.397

0.177

PIRPIRACETACETEtOHEtOH

ACAC

aaaa

aaaa

aaaa

ATP ADPATP ADP

RNARNA

OO22EE OO22

COCO22 COCO22EE

LIPLIP


FUMFUM AKGAKG

SUCCoASUCCoASUCSUC

MALMAL ISOCITISOCIT

OACOAC

SODSOD

SODSOD

SODSOD

PROTPROT

PROTPROT

PROTPROT

6.122

1.470

8.850

3.564

0.079

8.988

0.025

0.121

0.102

0.166

0.097

0.023

0.069

0.138

0.137

4.130 4.267

0.247

0.017

0.004

0.022

NHNH44EE NHNH44

0.724nunu

0.174

aaaa

0.063

0.014

0.046

1.470

1.470

1.470

1.345

1.3491.349

1.397

1.397

RATIO P-/P+ GLUC

RNARNA


44/64

GLUCGLUC

GLUC6PGLUC6P

FRUC6PFRUC6P

3PG3PG

GAPGAP

PYRPYR

PEPPEP

ACETACETEtOHEtOH

ACAC

RIBU5PRIBU5P


GAPGAPSED7PSED7P

FRUC6PFRUC6P

aaaa

aaaa

aaaa

aaaa

aaaa

E4PE4P

CARBCARB

RNARNA

COCO22 COCO22EE

0.92

0.99

1.23

LIPLIP


MALMAL ISOCITISOCIT

OACOAC

RNARNA

GLYCGLYC

PROTPROTPROTPROT

PROTPROT

PROTPROT

1.23

1.23

1.60

1.38

1. 8 2 ( 1

. 3 9 )

4.49

3.34

1.82(1.46)

1.60

1.39

1.60

0.36

1.23

3.73

1.00 1.09

1.60

1.63 1.82

1.80

1.84

1.74 1.05

1.40

1.82(1.16)

1.82(1.60)1.82(1.60)

1.82(0.96)

1.11

1.11

1.11

NHNH44EE NHNH44

1.33

LIPLIP

4 . 4 9

PROTPROTaaaa

RNARNA

nunu

nunu

1.32

nunu

1.10

aaaa

1.46

1.82(1.41)

1.60

1.60

1.60

1.61

1.61

1.80

RNARNA SODSOD

P+P+ : GLUC, EtOH-Exp.

, EtOH-Stat.


45/64

GLUCGLUC

GLUC6PGLUC6P

FRUC6PFRUC6P

3PG3PG

GAPGAP

PYRPYR

PEPPEPACETACETEtOHEtOH

ACAC

RIBU5PRIBU5P


GAPGAPSED7PSED7P

FRUC6PFRUC6P

aaaa

aaaa

aaaa

aaaaaaaaE4PE4P

CARBCARB

COCO22 COCO22EE

LIPLIP

AcCoAAcCoAmitmit

AcCoAAcCoAcitcit

MALMAL ISOCITISOCIT

OACOAC

SODSOD

SODSOD

SODSOD

SODSOD

PROTPROTPROTPROT

PROTPROT

PROTPROT

NHNH44EE NHNH44

LIPLIP

PROTPROTaaaa

RNARNA SODSOD

nunu

aaaa

RNARNA

nunu

GLYCGLYC

AcCoAAcCoA

3.844

-0.250

-0.246

4.169

0.0000.000

6.256

-0.449

0.626

6.122

0.043

0.577

1.470

0.000

0.526

8.850

1.774

2.476

0.079

0.033

0.008

0.102 0.047 0.079

0.166 1.836 0.6080.069 0.028 0.009

0.029

0.0100.007

0.138

0.043

0.051

0.208

0.065

0.027

2.232

0.218

-0.780

0.105

0.029

0.043

0.137 1.826 0.598

4.130-1.826

-0.598 4.267

0.000

0.000

0.029

0.010

0.007

0.234

0.075

0.082

0.325

0.109

0.137

0.177 0.060 0.072

0.148

0.050

0.065

0.559

0.185

0.219

4.611

0.000

0.000

0.247

0.000

0.000

0.048 0.020 0.005

0.025 0.010 0.002

0.006

0.003

0.001

0.022

0.006

0.001

0.042

0.012

0.002

0.019

0.006

0.001

0.724

0.221

0.221

0 . 0 0 2

0. 0 0 1

0. 0 0 2

0.174

0.053

0.046

0.057

0.015

0.011

0.063

0.021

0.021

0.046

0.019

0.007

1.470

1.470

1.158

0.956

1.397

1.7421.001

0.000

1.668

0.394

0.000

0.531

0.000

0.014 0.000 0.025

0.006

0.000

0.007

0.000

0.140

0.109

6.151

-0.478

0.584

0.177 0.060 0.072

0 . 0 2 5 0 .0 0 0 0 .0 3 1

0.017 0.000 0.030

0.028 0.000 0.035

0.004 0.000 0.004

0.004

0.000

0.004

0.025

0.010

0.002


46/64

eglu

etoH

coseglu

etoH

coseglu

etoH

cosP-


47/64

P-

EtOH/Gluc

Glucose Ethanol

Central metabolic

pathway

82 genes


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Discrete mathematical modelsapplied to genetic regulation and

metabolic networks


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Microarrays

MetabolicFlux

Analysis

Gene network

Metabolic network

Models

Traditionaltechnologies


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Phenomena to model

Genetic and metabolic

adaptation of E. coli to 3different carbon sources

Substrates: Glucose, Glycerol

and AcetateGlycolysis and TCA

8 possible substrate combinations

8 Phenotypes

Phenomena has beendescribed using Microarrays

(MA) and Metabolic Flux

Analysis (MFA)

Genes regulando el


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Genes regulando el

metabolismo

0 Inactivo

1 Activo

1 / 2 / 3 Activo

0

1 / 2 / 3

Estados

Señales = Biochemicals / Reguladores

-1 / -2 / -3

Flujo Metabólico de Enzima

-1 Inactivo

Gen

Signal2 GeneSignal1

EnzComp B1 Enz1

Enz2 /

Signal2Signal

Enz1 /

Signal1

Estudio de dinámica


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Estudio de dinámica

del modelo

67 nodos

28 genes21 enzimas

18 reguladores / compuestosbioquímicos

Reguladores Ficticiospara que modeloalcance Fenotipos

AlgoritmoDefinir combinación de sustratos

Generar 105 vectores aleatorios

Actualizar en forma paralela

Alcanzar atractor


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Atractor = Phenotype

Each atractor of model

corresponds to a phenotype

Atractors are very stable

Similarity between Atractorswith:

Glucose present

Glycerol present and Glucose

absentOnly Acetate present and All

others absent


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Cultivo de Tejidos

- tejidos

- células (e.g. sanguíneas)

- órganos


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Células para Terapia Celular

Vectores para Terapia Génica


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Terapia Génica

• Alcoholismo

• Osteoporosis

• Parkinson• Cancer (e. mama - gene BRCA-1)

• Artritis

• Hemochromatosis

• Alzheimer


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Vector de Primera Generarión

Vector de Tercera Generación o “gutless”


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Multiproduct and multihost batchplant

Fermentation

Microfilter

Homogenizer

Sterile filtration

Ultrafilter

Chromatography

Centrifuge

Bead mill Microfilter

IB SolubilizationDiafiltration Sulfonation

RefoldingUltrafiltration diafiltrater

Centrifuge

ChromatographyChromatography

Ultrafiltration diafiltrater

All the hosts

Yeast intracellular and E. coli

E. coli only

E. coli and CHO cells

OPTIMAL PROCESS SYNTHESIS FOR THE PRODUCTION OFMULTIPLE RECOMBINANT PROTEINS

Iribarren, Montagna, Vecchietti, Andrews, Asenjo and Pinto


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Documents

ac citrico.pdf