Bacterias para el medio ambiente: Bacterias para el medio ambiente: de la Bioremediación de la Bioremediación a la Biología Sintética a la Biología Sintética Víctor de Lorenzo Víctor de Lorenzo Centro Nacional de Biotecnología Centro Nacional de Biotecnología Madrid ( Madrid ( Spain Spain ) )
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Bacterias para el medio ambiente: Bacterias para el medio ambiente:
de la Bioremediación de la Bioremediación
a la Biología Sintéticaa la Biología Sintética
Víctor de LorenzoVíctor de Lorenzo
Centro Nacional de Biotecnología Centro Nacional de Biotecnología
Madrid (Madrid (SpainSpain))
La Cloaca Maxima (Roma, s. V adC)
Los tres dominiosdel árbol de la vida
LUCA
Microbial properties of environmental interest
• Biodegradative pathways for recalcitrant compounds
• mineralization of toxic pollutants
• removal or organic sulphur from soil
• biosensors
• green chemistry
• Tolerance to heavy ions
• volatilization
• bioavailability/toxicity assays
• bioaccumulation:removal of toxic ions
• biotransformation and re-speciation
• Surfactant production: bioremediation of oil spills
BIO-REMEDIACION
ATóxico
BIO-REMEDIACION
A BTóxico Inocuo
BIO-REMEDIACION
A BTóxico Inocuo
Catalizador biológico
BIO-REMEDIACION
A BTóxico Inocuo
Catalizador biológico
BIO-REMEDIACION
B
CO2 + H2Omineralización
C transformación
B inmovilización
• Natural attenuation
• Bioaugmentation
(incl. genetic engineer.)
• Biostimulation
(Eco-engineering)
Strategies in Bioremediation
Our Our experimental experimental systemsystem……
CH3
Cl
Cl
Cl
Cl
Cl
NO2
CH3
(CH)n
CH3
OH
CH3
Cl
CH3
NO2
NH2
CH3
OH
Enrichment cultures
Accessing the diversity of soil microbes
Cepa (Plásmido) Contaminante
P. putida mt-2 (pWW0) Tolueno
Pseudomonas sp, CF600 (pVI150) Fenol
Burkholderia sp. RP007 Fenantreno
P. putida TMB Trimetilbenceno
P. putida NAH (NAH7) Naftaleno
Acinetobacter sp. ADP1 Aril esteres
R. eutropha (pJP4) 2,4 D
Pseudomonas sp. (pP51) Triclorobenceno
P. putida UCC22 (pTDN1) Anilina
P. oleovorans (pOCT) n-alcanos
Burkholderia cepacia LB400 Bifenilo, PCBs
Pseudomonas sp. VLB120 Estireno
CH3 COOH OH
OH TCA
Xyl UWCMABN
Upperoperon Xyl XYZL
toluatedioxygenase
Xyl TEGFJQKIH
C2,3O et alLower (meta)
operon
El sistema TOL de Pseudomonas putida
CH3 COOH
Xyl UWCMABN
Upperoperon
CH3 CH2OH
De la biodegradación a la química verde
COH COOH
xylA xylB xylC
CH3 CH2OH
De la biodegradación a la química verde
xylA
CH CH2CH=CH2
xylA
O
estireno epóxido
microorganismos
(156 k)
ecosistemas
Diversidad conocida
plant
(270 k)
animal
(1.230 k)
microorganisms
200 k
ecosystems
plants(250k)
animal1500k
animalsplants
microbes
human
known existing
fungi
80 k
protista
100 k
known
eubacteria & archaea
existing
nonculturable
culturable
10 k
Exploring new worlds…
Metagenomics
16S RNA
DGGE analysis of 16 s RNAs of lindane-polluted soils
enriched
Acceso al metagenoma microbiano
Extraction of intact bacterial fraction from soil
Fractionation of metagenomicsoil DNA for cloning in lambda vectors
Pero… ¿podemos mejorar
a la Naturaleza?
¡Si! pero utilizando sus
mismos mecanismos
Declorination activity of linA-like clones
0,000
0,200
0,400
0,600
0,800
1,000
1,200
1,400
5F0
9
10
H0
8F0
5
5H
09
8A
0
3C
04
1E
07
2A
0
8E
12
4D
0
3G
11
5G
04
9A
0
8C
09
5G
11
clone
Ac
tiv
ity WT
Act
LinA geneMutagenic
PCR
ScreenActivity assay
DataAnalysis
Cys -> Arg
Study ofImprovedVariants
Next round
DIRECTED EVOLUTION OF Lindane Dechlorinase (LinA)
plac
Reintroducing linA-in the
biodegradation network with
transgenic plants
José Eduardo González-Pastor, Carolina González de Figueras y
Víctor de Lorenzo
Centro de Astrobiología (INTA-CSIC) (Madrid)
Centro Nacional de Biotecnología (Madrid)
pCAMBIA3500, a binary vector to transform
Arabidopsis thaliana
• Replication origins for Escherichia coli and for Agrobacterium tumefaciens
• T-DNA from Agrobacterium (DNA that is excised and integrated in the plant) was
engineered to contain:
– a 35S promoter from cauliflower mosaic virus (CaMV35S) driving a gene encoding
resistance to phosphinothricin (herbicide) for selection of the plant transformants
– Two CaMV35S promoters driving expression of the transgenes, and a CaMV polyA
(modified by Carlos Alonso-CNB)
T-border
(left)
T-border
(right)
CaMV
polyA
CaMV
polyA
CaMV
35S
2x
CaMV
35S
linA
phosphinothricin
linA-transgenic Arabidopsis thaliana growing in thepresence of lindane (10 m M)
linA control linA (C-His) control
70s
80s
90s
2Ks
Widening of Biotechnologies
Health
Food
Environment
ChemistryNanotech
?
Biología Sintética
Rediseñar los sistemas vivos
The big leap forward: from GeneticEngineeringto SyntheticBiology
Catalog of parts
Bacterial promoters as logic gates (I)
Bacterial promoters as logic gates (II)
xylUWCMABNPu Pm xylXYZLTEGFJQKIH xylS Ps Pr xylR
Upper operon meta operon
benzoate
m, p-xylene
The regulation of the TOL system
toluateCH3
R
COOH
R
Regulation of the TOL system as a whole of logic gates
What can be done with allthis?
Engineering developmental patterns in bacterial layers
by designing genetic circuitswith Quorum-Sensing components
R. Weiss
Programming anartificial genetic circuit with parts ofthe bacterialQuorum Sensingsystem
Designing E. coli strains withLight-responsive properties
Voigt
Chimaeric light receptor
• Phytochrome Cph1 from Synechocystis• EnvZ• Phycocyanobilin (PCB) ho1, pcyA
Light-dependent lacZ expression
Mercury vapor lamp
Projected image
Double Guass
focusable lens
632nm
bandpass fi lter35mm slide
Only games?
H H H H H H
H
H H H H H
H
H
H
H
H
H
H
H
Cl Cl
Cl Cl
Cl Cl
H H H H H
H
H H H H
H
H
H
+ H+ + Cl-
+ H+ + Cl-
+ H+ + Cl-
[e- don or] ox
[e- don or]red Pd(0)cell
Pd(II) sol
H
Pd(0)cell
Pd(0)cell /H2 [e- don or]red
[e- don or]ox
S. oneidensi s a
b
c
Dechlorinating PCBs with Bio/inorganic catalysts
G6P
Glucose
PYRAcCoA
Acetate
Isocitrate
-Ketoglutarate
SuccinateGlyoxylate
Fumarate
Malate
Oxaloacetate
LactateEthanol
GlucoseG6P
PEP
PYR
ATP ADP
CO2
Formate
Formate
(Secreted) H2
NAD
H
NAD
Citrate
Pentose
Phosphate
Pathways
Building abetter H2
producerBuilding a new
chromosome based
on genome
sequencesMaximizing
conversion
to H2
Maximizing
renewable
resource
utilization
Towards a an H2 super-producer (?)
2006 2008 2010 2012 2014
Synthetic
microbial core Functional
genomics core
Computational
microbial design lab Protein
complex core
Genetic circuit
repository
Microbial Fuel Cell
R
I
Transcriptional regulators à la carte?
Antb reporter
Epitope presentation
Enzymatic activity
Optical output
R
Circuitdesign
P
Area Reduction:THE DISTRIBUTED & SPECIALIZED SENSOR
The MMDS provides a distributed & specialized sensor thatdetects explosive traces and reacts providing an enhanced EMevidence.
A second sensor (a mobile one) easily detects the new EMevidence, which is the translation and amplification of theoriginal explosive one.