[PPT] - i c r o g u a r d s OVERVIEW The m i c r o g u a PowerPoint Presentation

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LGE UNICAMP

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BIOFUELS

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ETHANOL

OVERVIEW

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Costs-benefits

Ideal conditions

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Source: www.bioetanoldecana.org - Adapted

Land use in Brazil

Total land area (851 Mha, 100%) Agricultural properties area (355 Mha, 42%) Cropland area (76.7 Mha, 9%) Sugarcane cropland for energy-use area (3.6 Mha, 0.5%)

OVERVIEW

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Raw Material Energy

Produced/Utilized

Avoided emissions Sugarcane 9.3 89% Corn 0.6 – 2.0

30% to 38%

Wheat 0.97 – 1.11 19% to 47% Beet 0.97 – 1.11 35% to 56% Cassava 1.6 – 1.7 63%

Source: www.bioetanoldecana.org

Raw material utilized to produce ethanol

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Source:Statistical Review of world energy 2009 - Adapted

Global ethanol production

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Source: D.L. Gazzoni, Adapted

Production and costs of ethanol in Brazil

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ETHANOL

A lot can still be done!

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Good substitutes for

il-based fuels

Solve the environmental problems

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Biofuels

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Biofuels do not compete with food and land use

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Biofuels versus Food

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Enable appropriate land use

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Scientific research Should continue - obtain more efficient biofuels using genetic engineering

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ETHANOL

Project

Costs Land use

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How is ETHANOL produced in Brazil?

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How is ETHANOL produced in Brazil?

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CONTAMINANTS

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How is ETHANOL produced in Brazil?

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iGEM project - CONTAMINATION

YEAST

Ethanol Insulin, yogurt

BACTERIA

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… Prepare yourselves to get into the microguards´ lives

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RECOGNITION SYSTEM KILLING SYSTEM

The Yeastguard OVERVIEW

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First Mechanism (increased lactate sensibility) Second Mechanism (recognizing lactate on the interior of the cell)

Biobrick submitted! Biobrick submitted! Recognition system

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RECOGNITION SYSTEM KILLING SYSTEM

The Yeastguard OVERVIEW

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Biobrick submitted! killing system

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RECOGNITION SYSTEM KILLING SYSTEM

Yeastguard OVERVIEW

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Successful assembled biobrick

 BBa_K284002: JEN1 Promoter  BBa_K284003: Partial DLD promoter  BBa_K284023: EYFP regulated by ADH1 promoter (characterization device)  BBa_K284001: Lysozyme from Gallus gallus  BBa_K284016: Lysozyme constitutive expression (characterization device)  BBa_K284017: Lysozyme under control of DLD promoter (characterization device)

THEYeastguARd

results

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Recognition system Differentiation system Killing system

The coliguard OVERVIEW

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Briefly,

If contaminants conjugate with our coliguard: 1. Induces Py 2. Releases AI2 3. Activate Differentiation and Killing mechanism If contaminants release AI2 themselves: 1. . 2. . 3. Activate Differentiation and Killing mechanism

skip steps! Recognition system

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Recognition system Differentiation system Killing system

The coliguard OVERVIEW

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Worker Cells Killer Cells Amount Amount Basal proportion!

Differentiation system

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Absence of contaminants:

Differentiation system

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Presence of contaminants:

Differentiation system

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Differentiation system

Presence of contaminants:

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Differentiation system

Elimination of contaminants:

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Absence of contaminants:

Differentiation system

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Worker lineage characteristics!

The slippage mechanism controlling the basal proportions

(AGTC)10 Differentiation system

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The slippage mechanism controlling the basal proportions

(AGTC)10 (AGTC)9 Killer lineage characteristics! Differentiation system

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Integration between the mechanisms of slippage and Cre-Recombinase

Slippage error doesn’t occurs (most cases):

Cell Cycle! Conjugation Inhibition System Worker lineage Differentiation system

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Killer lineage

Integration between the mechanisms of slippage and Cre-Recombinase

Slippage error occurs (few cases):

(AGTC)9 Differentiation system

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Enhanced Amount of Killers cells induced by contaminants

Differentiation system

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Recognition system Differentiation system Killing system

The coliguard OVERVIEW

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Killing system

The Kamikaze System The Colicin System

CeaB lethal gene (colicin E2) into F plasmid CeiB antidote gene into genomic DNA

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Recognition system Differentiation system Killing system

The coliguard OVERVIEW

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Successful assembled biobrick

 BBa_K284008: Py promoter + RFP device (characterization device)  BBa_K284031: Cre-Recombinase without ATG start codon  BBa_K284022: T4 endolysin under control of T7 promoter (characterization device)  BBa_K284022

THE COLIGUARD

REsults

Characterized parts

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The coliguard

results

Characterizing BBa_K284022

 Transformations into E. coli strain C43 (T7 promoter is induced by IPTG)  Grown inocula were diluted to starter OD=0,2  Once OD=0,8 was reached  induction with IPTG  Incubation for 4 hours at 37⁰C

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Characterizing BBa_K284022

The coliguard

results

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Characterizing BBa_K284022 Plated each culture into solid LB-AMP media

The coliguard

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Characterizing BBa_K284022 SDS-PAGE

The coliguard

results

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Summary  2 contamination control models designed, one for prokaryotes and one for eukaryotes  30 new biobrick parts and devices designed  9 new biobrick parts and devices constructed and submitted to the registry  4 new biobrick parts or devices were tested  1 new biobrick device worked as expected (BBa_K284022)  An existing Biobrick part was characterized (BBa_K112806)  A new approach to an issue of Human Practice in synthetic biology as it relates to

ur project was outlined and detailed

Accomplishments

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This work could not be done without the sponsor of

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Special Acknowledgements:

Prof. Dr. Fernando Costa. Dean, UNICAMP.
Prof. Dr. Luís Cortez. CORI, UNICAMP.
Prof. Dr. Fábio Papes. Dept. Genética, Evolução e Bioagentes, UNICAMP.
Prof. Dr. Paulo Arruda. Dept. Genética, Evolução e Bioagentes, UNICAMP.
Prof. Dr. Gonçalo A. G. Pereira. Dept. Genética, Evolução e Bioagentes, UNICAMP.

Support:

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thank you!!!!