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Preparing a cyanobacterial chassis for H2
production: a synthetic biology approach
Catarina Pacheco
Cell and Applied Microbiology Group
IBMC, INEB
E4. Genómica funcional e biologia sintética
Encontro Nacional de Ciência - Ciência 2009
Fundação Calouste Gulbenkian,30th July 2009
Synthetic Biology is...
... the design and construction of new biological parts,
devices and systems and the re-design of existing, natural
biological systems for useful purposes.
Synthetic Biology is the application of engineering
concepts to biology
Standardized
parts
Assembly of modules
and circuits
Incorporation in
a chassis
BioBrick™ parts assembly
BioBrick™
Standardized DNA fragment
designed
for
a
specific
purpose and that can be easily
assembled with other bricks
to generate modules and
devices.
e.g.
promoter
sensor
modified gene
http://partsregistry.org/
Chassis
“The candidates for chassis should be well studied organisms with
high throughput genomic and proteomic data available, minimalist in
terms of the subset of genes that will allow retaining viability, and
easy to engineer with the available molecular tools, becoming a
versatile platform for multiple purpose applications”
Escherichia coli
Yeast
Bacillus subtilis
Jan.07- Jan.10
Consortium members:
Instituto de Biologia Molecular e Celular (Portugal)
École Polytechnique (France)
Universidad Politécnica de Valencia (Spain)
Uppsala Universitet (Sweden)
University of Sheffield (UK)
Weizmann Institute of Science (Israel)
FP6-2005-NEST-PATH
Contract no.: 043340
BioModularH2
Design
In silico analysis
Computational design of parts
and modules
Construction
Synthesis of parts
Assembly of modules
Preparation of the chassis
Caracterization
Caracterization of parts/modules
Incorporation in the chassis
Evaluation of the final product
Final goal
A
cyanobacterial
chassis
that
together with the designed devices
will harvest solar energy for H2
production.
The synthetic parts and modules
will be available for other
biotechnological applications
Photoautotrophic chassis - Synechocystis sp. PCC 6803
 the most studied cyanobacteria
 unicellular and non-N2-fixing
 simple nutritional requirements
 naturally transformable
 molecular tools for manipulation available
 small genome comprising a 3.6 Mb genome
and 7 plasmids (1st cyano genome sequenced)
Preparation of the chassis
Tuning respiration
Oxygen sensing
Oxygen
consumption
Native
hydrogenase (s)
etc…
Nuclease(s)
Highly-efficient
O2-tolerant hydrogenase
•
Reduce constraints, e.g. enhance transformation efficiency
•
Remove redundant genes / parts
•
Minimize O2 production / maximize O2 consumption  H2ases are very sensitive to O2
Deletion of redundant parts –
generation of a hydrogenase deficient mutant
hoxY
hoxH
Possesses hoxYH
Sensitive to kanamycin
Resistant to sucrose
Deletion of hoxYH
Resistant to kanamycin
Sensitive to sucrose
Lacks hoxYH
Sensitive to kanamycin
Resistant to sucrose
Hydrogen Producing Device (HPD)
HydA1_Fd
Hydrogenase module
Fe-only hydrogenase fused to ferredoxin –
Chlamydomonas reinhardtii
Maturation module
HydEF + HydG – Chlamydomonas reinhardtii
Homology models based on Chang et al.
2007 (Biophys J, 93:3034-45)
Identification of neutral sites for the insertion of
synthetic modules
Genes encoding proteins:
- unknown or hypothetical
- with maximum length of 300 a.a.
- without predicted transmembrane domains (TMHMM Server v. 2.0)
- primary or secondary structure without relevant homologues
- that do not interact with other proteins in two-hybrid system
(CyanoBase data)
16 potential neutral sites identified
Analysis of gene expression by RT-PCR
Generation of deletion mutants
in the ORFs corresponding to
the neutral sites N5, N7, N8,
N10, N15 and N16.
Mutant analysis will reveal the true
neutral sites that can be used for
the integration of synthetic
modules and devices.
Design and characterization of parts for H2 production
Oxygen Consuming Device (OCD)
SINGLE-protein modules
O2  H 2 O
- A-type flavoprotein (ATF) – Synechocystis sp. PCC 6803
- Laccase – Escherichia coli
TWO-protein module
O2  H2O2  ½ O2 + H2O
- Glucose oxidase – Penicillium amagasakiense
+
Catalase – Synechocystis sp. PCC 6803
Testing the expression of A-type flavoprotein (ATF) module
in Escherichia coli
Constructions used in the test:
Wild-type
MW
-
+
T9002 *
-
+
Promoterless
LuxR controled
ATF **
ATF ***
+
-
+
-
AHL
100 kDa
T9002 *
75 kDa
PtetR
luxR
PluxR
gfp
ATF
50 kDa
(63 kDa)
Promoterless ATF **
PtetR
luxR
37 kDa
gfp
ATF
LuxR controled ATF ***
PtetR
luxR
PluxR
ATF
gfp
25 kDa
GFP
(27 kDa)
- A synthetic module that can be used
for the controlled expression of the
ATF was obtained.
The Cellular and Applied Microbiolgy group
Thank you for your attention