MEXICO CITY 20 million people

Land area of 1,485 km

We have a BIG problem…

MEXICO CITY 20 million people

Land area of 1,485 km

We produce 35 Ton of CO2/year

We have a BIG problem…

Problem 1: Polution in

México

Problem 2: Fuels are

running out.

Plants

Algaes

E. coli

Hey guys, have you

heard about PNSB?

E. coli

INPUT

OUTPUT

Photosynthesis

O2

iGEM Team

Butanol Production Pathway

Butanol

…

Rhodofactory:

Controlling genetic expression, an oxygen and light response

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

Measurement Kit

pSB1C3 C R

Promoter selection iGEM contribution

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

What if we could introduce this Regulatory Systems in

another bacteria?

Will they work…?

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.

Modeling

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

Experimental Approach

How we did it?

Experimental Approach

• BioBricks assembly.

Constructs

• Biparental

• Triparental

• pRK415

Conjugation • Flow Cytometry

• Fluorescence microscopy

Characterization

Results

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.

Results

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

• 2 New dependent Promoters for PNSB.

• 2 Functional Biobricks

Devices.

Conclusions

Perpectives

HUMAN PRACTICES

Using QFD To identify priorities for biosecurity in México

Biosafety Report

Biosafety Report

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

Creating collaboration networks

iGEM Meet up!

Encouraging new teams!

iGEM Experience

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

And our Sponsors:

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!!!!