MEXI XICO CO CI CITY TY Land area of 1,485 km 20 million people We have a BIG problem …
MEXI XICO CO CI CITY TY We produce e 35 Ton of CO 2 /ye year ar Land area of 1,485 km 20 million people We have a BIG problem …
Problem 1: Polution in México Plants Algaes E. coli Problem 2: Fuels are running out.
Hey guys, have you heard about PNSB? E. coli
INPUT iGEM Team Butanol Production Pathway OUTPUT O 2 Butanol Photosynthesis …
R hodofactory: Controlling genetic expression, an oxygen and light response
Regulatory PpsR Network AppA AppA PpsR Photosynthetic Membrane components Electron P Transport PrrA Chain Substrates PrrA O 2 H 2 O P Prr B
Our BioBricks: Light and Oxygen system RBS Prom GFP DT B0030 PpsR B0014 BBa_K776018 RBS AppA Prom GFP DT RBS Prom RBS PpsR B0030 DT PpsR B0014 B0030 J23104 B0030 B0014 BBa_K776020
Light and oxygen system O 2 AppA AppA sense light PpsR is No complex and O 2 oxidized formation AppA PpsR PpsR Inactive AppA can’t bind PpsR Repression by PpsR PpsR Prom RBS AppA Prom GFP DT RBS RBS PpsR J23104 B0030 DT PpsR B0014 B0030 B0030 B0014 BBa_K776020 No GFP expression
O 2 O 2 Light and oxygen system AppA sense light and O2 PpsR is reduced AppA Complex PpsR PpsR AppA PpsR formation AppA Active binds PpsR No repression of PpsR RBS AppA Prom GFP DT Prom RBS RBS PpsR B0030 DT PpsR B0014 J23104 B0030 B0030 B0014 BBa_K776020 GFP Expression is induced.
Our BioBricks: Oxygen system RBS Prom GFP DT B0030 PrrA B0014 BBa_K776019 RBS RBS RBS PrrA PrrB DT RBS DT Prom GFP Prom PrrC B0030 B0030 B0030 B0030 B0014 B0014 J23104 PrrA BBa_K776021
Oxygen system O 2 Electron chain transport No-Autopshorylation P Redox PrrB Inactive form PrrA PrrC signal No transcription RBS RBS RBS PrrA PrrB DT RBS DT GFP Prom PrrC B0030 B0030 B0030 B0030 B0014 B0014 Prom PrrA J23104 BBa_K776021 No GFP expression
O 2 O 2 Oxygen system Phosphate group is Autophosphorylation transfered P P Redox PrrA Prr B PrrC signal Transcription Activation P PrrA RBS RBS RBS PrrA PrrB DT RBS DT GFP Prom PrrC B0030 B0030 B0030 B0030 B0014 B0014 Prom PrrA J23104 BBa_K776021 GFP Expression is induced.
Promoter selection ? RBS RBS RBS PrrA RBS PrrB DT DT Prom GFP Prom PrrC B0030 B0030 B0030 B0030 B0014 B0014 PrrA
Promoter selection iGEM contribution pSB1C3 C R Measurement Kit
GFP expression 6E+14 MK J23101 5E+14 GFP relative expression MK J23102 4E+14 MK J23104 3E+14 MKJ23107 MK J23108 2E+14 MK J23111 1E+14 Mk J23115 0 0 10000 20000 30000 40000 50000 60000 70000 Time (sec) Promoters relative strenght 30 25 J23102 J23104 20 J23107 J23104 RPUs 15 J23108 J23111 10 J23115 5 0 1 Promoters
What if we could introduce this Regulatory Systems in another bacteria? Will they work…?
Evaluate the modularity of regulatory systems Applying Synthetic Biology 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 Aromatic Biofuel compounds Production Degradation Indol Production Metal Oxides Detoxification Hydrogen Vitamin B12 Production Production Carotenoid Production PHB Production
What was the plan? Source microrganism R. sphaeroides. New target microorganism Rhodopseudomonas palustris.
Modeling
Rule-based model Protein-Protein interaction Concentration of our main proteins Interactions Parameters Conditions network Rate constant Description Time of reduction of efc3(ms) Quinone Pool PpsR reduction O 2 kprplus(mV) mediated by AppA k2 (M -1 .s -1 ) Trimer formation k3 (M -1 .s -1 ) Complex formation Heme cofactor kp1 (M -1 min - O 2 binding to through C- 1 ) terminal domain Transphosphorylation kp3 (s -1 ) of PrrB and PrrA k1(M -1 .s -1 ) Complex dissociation
Light and oxygen system O 2 Rhodobacter spharoides Rhodopseudomonas palustris 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
Oxygen system O 2 Rhodobacter spharoides Rhodopseudomonas palustris Time (hrs) Time (hrs) Graphic 2. Oxygen system simulation; anaerobic/ light conditions ; concentration of PrrA inactive/active PrrA inactive (not phosphorylated) PrrA active (phosporylated)
Experimental Approach
Experimental Approach How we did it?
Experimental Approach Conjugation • BioBricks • Flow Cytometry assembly. • Fluorescence • Biparental microscopy • Triparental • pRK415 Constructs Characterization
Results
O 2 Light and oxygen system Aerobic-Darkness R. sphaeroides 40.66% 45.00% PpsR promoter 40.00% 35.00% 30.00% 26.56% % GFP+ Bacteria 25.00% 21.21% 17.66% 20.00% AppA/PpsR system 11.91% 15.00% 10.00% 5.00% 1.38% Anaerobic-light 0.00% 1 2 PpsR Promoter AppA/PpsR system PpsR Aerobic/Darkness Anaerobic/Light Anaerobic/Darkness promoter AppA/PpsR system RBS Prom GFP DT B0030 PpsR B0014 RBS AppA Prom GFP DT Prom RBS RBS PpsR DT B0030 PpsR B0014 B0030 J23104 B0030 B0014
Oxygen system O 2 R. sphaeroides 8.00% 6.61% 7.00% Anaerobic/Light 6.00% % GFP+ Bacteria 5.00% 4.53% 3.96% 4.00% 3.00% 2.64% PrrA promoter 2.00% 0.58% 1.00% 0.62% 0.00% 1 2 PrrA Promoter PrrA/PrrB system PrrA/PrrB system Aerobic/Darkness Anaerobic/Light Anaerobic/Darkness RBS Prom GFP DT B0030 PrrA B0014 RBS RBS RBS PrrA PrrB DT RBS Prom GFP DT Prom PrrC B0030 B0030 B0030 B0014 B0030 B0014 J23104 PrrA
What was the plan? Source microrganism R. sphaeroides. New target microorganism Rhodopseudomonas palustris.
Results
O 2 Light and oxygen system R. palustris 60.00% Anaerobic/Light 51.40% 50.00% 40.00% PpsR 31.89% promoter % GFP+ Bacteria 30.00% 20.00% 16.90% 10.00% 2.70% 0.00% AppA/PpsR 1 2 PpsR Promoter AppA/PpsR system system Aerobic/Darkness Anaerobic/ Light Anaerobic/ Darkness RBS Prom GFP DT B0030 PpsR B0014 RBS AppA Prom GFP DT RBS Prom RBS PpsR DT B0030 PpsR B0014 J23104 B0030 B0030 B0014
O 2 Oxygen system R.palustris Anaerobic/light 35.00% 30.60% 30.00% PrrA promoter 23.99% 25.00% 22.80% % GFP+ Bacteria 20.00% 15.00% 10.00% PrrA/PrrB system 5.00% 2.51% 0.79% 0.49% 0.00% 1 2 PrrA Promoter PrrA/PrrB system Aerobic/Darkness Anaerobic/ Light Anaerobic/ Darkness RBS Prom GFP DT B0030 PrrA B0014 RBS RBS RBS PrrA PrrB DT RBS Prom GFP DT Prom PrrC B0030 B0030 B0030 B0014 B0030 B0014 J23104 PrrA
Summary Results Microorganism O 2 Light & Oxygen O 2 Oxygen Rhodobacter O 2 sphaeroides O 2 Light & Oxygen O 2 Rhodopseudomonas Oxygen palustris
Conclusions • 2 New dependent Promoters for PNSB. • 2 Functional Biobricks Devices.
Perpectives
HUMAN PRACTICES
Biosafety Report Using QFD To identify priorities for biosecurity in México
Biosafety Report
Integrate an interdisciplinary Educate legislators and committee to discuss IP and decision makers OS issues Create a solid IP Ownership report Structure for RSBP Establish a collaboration Inform scientist about the network between industries, advantages and disadvantages research centers and for IP and OS universities
Creating collaboration networks iGEM Meet up!
iGEM Experience Encouraging new teams!
How to create an iGEM team? Take the Go to the lab! Modeling decision Get Work in Prepare your institutional teams! results support Get a brilliant Human Fundraising idea Practices Expose your Project design idea
This year, 13 students took part in a 6 week-summer internship in the Systems and Synthetic Biology Lab (SSBL) 3 rd Summer Workshop in SynBio Preparing scientists for the future!
Instructors Paola Zárate-Segura PhD Agustino Martínez-Antonio PhD Researcher at Resarcher 3A, SNI I IPN.UPIBi-ESM Genetic Engineering Department, Bioprocess Department CINVESTAV Irapuato. Fernando Suaste-Olmos PhD Nuria Sánchez-Puig PhD Researcher at Institute of Cell Physiology Researcher at Institute of IFC-UNAM. Chemistry UNAM
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