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MEXICO CITY 20 million people
Land area of 1,485 km
We produce 35 Ton of CO2/year
We have a BIG problem…
Electron Transport
Chain
Substrates
Photosynthetic Membrane components
PrrA P
Prr
B
PrrA
P
PpsR
AppA
PpsR AppA
O2 H2O
Regulatory Network
Light and Oxygen system
Our BioBricks:
Prom PpsR
RBS B0030
DT B0014
GFP
BBa_K776018
BBa_K776020
RBS B0030
AppA Prom J23104
PpsR DT B0014
RBS B0030
Prom PpsR
RBS B0030
DT B0014
GFP
PpsR AppA
Pp
sR
No GFP expression BBa_K776020
AppA sense light
and O2
Inactive
O2
PpsR is oxidized
AppA can’t bind
PpsR
No complex formation
PpsR
AppA
Repression by PpsR
RBS B0030
AppA Prom J23104 PpsR DT
B0014
RBS B0030
Prom PpsR
RBS B0030
DT B0014
GFP
Light and oxygen system
Pp
sR
O2
AppA
No repression of PpsR
Complex formation
PpsR is reduced
AppA sense light
and O2
GFP Expression is induced.
Active
BBa_K776020
AppA binds PpsR
AppA PpsR
Pp
sR
O2
RBS B0030
AppA Prom J23104
PpsR DT B0014
RBS B0030
Prom PpsR
RBS B0030
DT B0014
GFP
Light and oxygen system
PrrA PrrB RBS
B0030 PrrC DT B0014
RBS B0030
Prom J23104
RBS B0030 Prom
PrrA
RBS B0030
GFP DT B0014
Prom PrrA
RBS B0030
DT B0014
GFP
BBa_K776019
BBa_K776021
Oxygen system
Our BioBricks:
PrrC PrrA Redox signal
No transcription
PrrB
P
Electron chain
transport No-Autopshorylation
Inactive form
PrrA PrrB RBS
B0030 PrrC DT
B0014
RBS
B0030 Prom
J23104
RBS
B0030 Prom
PrrA
RBS
B0030 GFP DT
B0014
No GFP expression BBa_K776021
O2 Oxygen system
PrrC PrrA
P Redox signal
Transcription Activation
PrrB
P
Autophosphorylation Phosphate group is transfered
PrrA PrrB RBS
B0030 PrrC DT
B0014
RBS
B0030 Prom
J23104
RBS
B0030 Prom
PrrA
RBS
B0030 GFP DT
B0014
PrrA P
GFP Expression is induced. BBa_K776021
O2 O2 Oxygen system
Promoter selection
PrrA PrrB RBS
B0030 PrrC DT
B0014
RBS
B0030
?
RBS
B0030 Prom
PrrA
RBS
B0030 GFP DT
B0014 Prom
0
1E+14
2E+14
3E+14
4E+14
5E+14
6E+14
0 10000 20000 30000 40000 50000 60000 70000
GFP
rel
ativ
e ex
pre
ssio
n
Time (sec)
GFP expression
MK J23101
MK J23102
MK J23104
MKJ23107
MK J23108
MK J23111
Mk J23115
0
5
10
15
20
25
30
1
RP
Us
Promoters
Promoters relative strenght
J23102
J23104
J23107
J23108
J23111
J23115
J23104
Applying Synthetic Biology
Evaluate the modularity of
regulatory systems
Generate a set of
regulatory BioBricks for
PNSB
Our New Chassis!
Rhodopseudomonas palustris Metabolic versatility
Aerobic Chemoheterotrophic growth Chamoautotrophic growth
Anaerobic
Photoheterotrophic growth Photoautotrophic growth
Our New Chassis!
Rhodopseudomonas palustris
Biotechnological potential
Biofuel Production
Indol Production
PHB Production
Aromatic compounds Degradation
Hydrogen Production
Vitamin B12 Production
Metal Oxides Detoxification
Carotenoid Production
Source microrganism R. sphaeroides.
What was the plan?
New target microorganism
Rhodopseudomonas palustris.
Rule-based model
Protein-Protein interaction Concentration of our main proteins
Parameters Interactions
network Conditions
Rate constant Description
efc3(ms) Time of reduction of
Quinone Pool
kprplus(mV) PpsR reduction
mediated by AppA
k2 (M-1.s-1) Trimer formation k3 (M-1.s-1) Complex formation
kp1 (M-1 min-
1)
Heme cofactor
binding to through C-
terminal domain
kp3 (s-1) Transphosphorylation
of PrrB and PrrA
k1(M-1.s-1) Complex dissociation
O2
O2
Graphic 1. Light and oxygen system simulation; anaerobic/light condition. Concentration of complex
formation (AP2), that allows GFP expression, over time.
+ Oxidized - Reduced
AP2 Complex formation
O2
Rhodobacter spharoides
Rhodopseudomonas palustris
Light and oxygen system
PrrA inactive (not phosphorylated) PrrA active (phosporylated)
O2
Rhodobacter spharoides
Rhodopseudomonas palustris
Graphic 2. Oxygen system simulation; anaerobic/ light conditions ; concentration of
PrrA inactive/active
Time (hrs) Time (hrs)
Oxygen system
Experimental Approach
• BioBricks assembly.
Constructs
• Biparental
• Triparental
• pRK415
Conjugation • Flow Cytometry
• Fluorescence microscopy
Characterization
Anaerobic-light
PpsR promoter
AppA/PpsR system
PpsR promoter
AppA/PpsR system
Aerobic-Darkness
17.66%
40.66%
1.38%
26.56%
21.21%
11.91%
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
40.00%
45.00%
1 2
% G
FP+
Bac
teri
a
PpsR Promoter AppA/PpsR system
R. sphaeroides
Aerobic/Darkness Anaerobic/Light Anaerobic/Darkness
Light and oxygen system O2
Prom
PpsR
RBS
B0030 DT
B0014
GFP
RBS
B0030
AppA Prom
J23104 PpsR DT
B0014
RBS
B0030
Prom
PpsR
RBS
B0030 DT
B0014
GFP
0.62%
3.96%
2.64%
4.53%
6.61%
0.58%
0.00%
1.00%
2.00%
3.00%
4.00%
5.00%
6.00%
7.00%
8.00%
1 2
% G
FP+
Bac
teri
a
PrrA Promoter PrrA/PrrB system
R. sphaeroides
Aerobic/Darkness Anaerobic/Light Anaerobic/Darkness
Anaerobic/Light
PrrA promoter
PrrA/PrrB system
O2 Oxygen system
PrrA PrrB RBS B0030 PrrC DT
B0014
RBS B0030
Prom J23104
RBS B0030
Prom PrrA
RBS B0030
GFP DT B0014
Prom PrrA
RBS B0030
DT B0014
GFP
Source microrganism R. sphaeroides.
What was the plan?
New target microorganism
Rhodopseudomonas palustris.
2.70%
31.89%
16.90%
51.40%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
1 2
% G
FP+
Bac
teri
a
PpsR Promoter AppA/PpsR system
R. palustris
Aerobic/Darkness Anaerobic/ Light Anaerobic/ Darkness
O2 Light and oxygen system
PpsR promoter
AppA/PpsR system
Anaerobic/Light
Prom
PpsR
RBS
B0030 DT
B0014
GFP
RBS
B0030
AppA Prom
J23104 PpsR DT
B0014
RBS
B0030
Prom
PpsR
RBS
B0030 DT
B0014
GFP
0.79%
23.99%
30.60%
22.80%
2.51%
0.49% 0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
1 2
% G
FP+
Bac
teri
a
PrrA Promoter PrrA/PrrB system
R.palustris
Aerobic/Darkness Anaerobic/ Light Anaerobic/ Darkness
O2
PrrA
promoter
PrrA/PrrB
system
Anaerobic/light
Oxygen system
PrrA PrrB RBS B0030 PrrC DT
B0014
RBS B0030
Prom J23104
RBS B0030
Prom PrrA
RBS B0030
GFP DT B0014
Prom PrrA
RBS B0030
DT B0014
GFP
O2
O2
O2
Microorganism Results
Rhodopseudomonas palustris
Rhodobacter sphaeroides
Light & Oxygen
Oxygen
Light & Oxygen
Oxygen
O2
O2
Summary
Integrate an interdisciplinary committee to discuss IP and
OS issues
Educate legislators and decision makers
Inform scientist about the advantages and disadvantages
for IP and OS
Establish a collaboration network between industries,
research centers and universities
Create a solid IP Structure for
RSBP Ownership report
Take the decision
Get institutional
support
Get a brilliant idea
Expose your idea
Project design
Fundraising
Work in teams!
Go to the lab! Modeling
Prepare your results
Human Practices
How to create an iGEM team?
This year, 13 students took part in a 6 week-summer internship in the
Systems and Synthetic Biology Lab (SSBL)
Preparing scientists for the future!
3rd Summer Workshop in SynBio
Agustino Martínez-Antonio PhD Resarcher 3A, SNI I
Genetic Engineering Department, CINVESTAV Irapuato.
Fernando Suaste-Olmos PhD Researcher at Institute of Cell Physiology
IFC-UNAM.
Paola Zárate-Segura PhD Researcher at
IPN.UPIBi-ESM Bioprocess Department
Nuria Sánchez-Puig PhD Researcher at Institute of
Chemistry UNAM
Instructors
We would like to thank all the people who helped us through our project and our panic attacks:
MBA .Gerónimo Villanueva For his outstanding support
Juan Carlos Martínez García PhD.
For his support in mathematical modeling.
Ing. Susana Ruiz. For her support in Molecular Biology Techniques.
Ana Lilia Hernández.
For her support in Molecular Biology Techniques.
Norma Silvia Sánchez Sánchez QFB, UNAM. For her advices and support.
Msc. Libertad Pantoja Hernández.
For her support in mathematical modeling.
Heliodoro Celis Sandoval PhD. For allow us to work in his Lab at IFC-UNAM.
Acknowledgments
Hector Silva-Gutierrez, Eduardo Moreno-Rodríguez, Cesar Villavicencio-Cordova. For their valuable collaboration.
Karen Nava Castro PhD.
For her advices and help in Flow Citometry.
Jorge Morales Montor PhD. For his support in the project and allow us to work in his lab at IIB, UNAM.
Jorge Vázquez Ramos PhD., Perla Castañeda López PhD., Raúl Garza Velasco QFB, Guadalupe
Castorena Adame QFB, Luz del Carmen Castellanos Román M.Sc., Rodolfo Pastelín Palacios PhD., Verónica Ramón Lic., Veronica Jazmin Sánchez Ortiz, Manuel Camacho Zaragoza, Angelica Montes
Trujillo, Javier De la Mora, Teresa Ballado, Alejandro "Buds". For helping us in our panic moments Ö
Abiel Treviño Garza
For his advices about wiki design.
Design: Ing. Alejandra Bartolo Gervacio
Ing. José Adolfo Martínez Olmedo Maria Luisa Saenz Jaramillo
Acknowledgments
More than 200 ligations
More than 80 DNA constructs
More than 800 cups of coffee
More than 500 digestions
More than 20 simulations
38 donated BioBricks
More than 600 geles More than 200 PCRs
More than 25 friday nights working at the Lab
More than 50 DNA samples lost
1 broken centrifuge. 1 burnt laminar flow cabinet
And all this just to be part of the iGEM experience 2012!!!!