MEXI XICO CO CI CITY TY Land area of 1,485 km 20 million - - PowerPoint PPT Presentation

MEXI XICO CO CI CITY TY

20 million people Land area of 1,485 km We have a BIG problem…

MEXI XICO CO CI CITY TY

20 million people Land area of 1,485 km We produce e 35 Ton of CO2/ye year ar 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

PrrB 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

PpsR

No GFP expression BBa_K776020 AppA sense light and O2 Inactive

O2

PpsR is

• xidized

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

PpsR

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 PpsR

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

1E+14 2E+14 3E+14 4E+14 5E+14 6E+14 10000 20000 30000 40000 50000 60000 70000

GFP relative expression

Time (sec)

GFP expression

MK J23101 MK J23102 MK J23104 MKJ23107 MK J23108 MK J23111 Mk J23115 5 10 15 20 25 30 1

RPUs 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

• f 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

% GFP+ Bacteria

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 % GFP+ Bacteria

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

% GFP+ Bacteria 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

% GFP+ Bacteria 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!!!!