WHAT IS TIRE MADE OF? POLICY AND PRACTICES

ENTREPRENEURSHIP

WHO WE ARE

REFERENCES

DEVULCANIZATION

RUBBER DEGRADATION

JET-FUEL PRODUCTION

MAIN CIRCUIT

ROXA AND LCP

11,05 tons of scrap tires were discarded since the first produced.

25% REINFORCED AGENT

WOMAN IN SCIENCE

Rubber BiotechnologiesWe are focused in a sustainable economy to input tire waste into a value chain.

Target MarketWe contacted our customers and planned our products according their needs in a short, mid and long term.

Instructors:Ana Paula Ulian AraujoRichard Charles Garratt

Advisors:Cristina KurachiJuliana Cancino-BernardiOtavio Henrique ThiemannValtencir Zucolotto

We submitted Lcp and RoxA as two new iGEM parts.

Polyisoprenes docking was impracticable in RoxA structure.

Predictions of Lcp structure pointed to rich disordered and low α-helix content.

High conserved Histidine amino acid can be a candidate to heme coordination in Lcp.

Students Members:Ana Laura de LimaBruno OnoCamila BramorskiCamila Maria S. BoralliCristiane Casonato MeloDanilo Kenji ZampronioDeborah Cezar MenconçaEverton SilvaFabiane Fantinelli FrancoGraziele Vasconcelos

Ivan Rosa e SilvaJennifer Machado SoaresKaren Freire CarvalhoLeticia Camargo TavaresLaís Canniatti BrazacaLaís RibovskiMariana Lopes GarciaPaula Maria Pincela LinsThiago MosqueiroVictor H. Rabesquine Nogueira

Business Model Pilot Plan

INTERVIEWS

VISITING PNEUMATIC INDUSTRY

SURVEY: Are there segregation and gender harassment in academia?

Meeting with National Association of Pneumatic Industry (ANIP)

HILDE HARB BUZZÁCurrent course: Doctorate at Biomolecular Physics

LETÍCIA ZAGOCurrent course: Master at Education of Physics

KRISSIA DE ZAWADZKICurrent course: PhD at Theoretical Physics)

NATHÁLIA BERETTA TOMÁZIOCurrent course: Master at Applied Physics

PYLADIES51% POLYISOPRENE

Polyisoprene chains are cross-linked with a reinforcing agent by a vulcanization process. Vulcanization confers hardness and resistance, making the tire more di�cult to recycle. Our process use a natural microorganism Acidithiobacillus ferrooxidans to perform rubber devulcanization in a simple manner.

We propose a safe circuit with RoxA and Lcp enzymes to potentialize polyisoprene degradation. An innovative exportation system attached to the enzymes was designed specifically to our chassis, Escherichia coli.

Figure 1. Left: Polyisoprene cross-linked bonds. Right: Flasks containing Acidithiobacillus ferrooxidans in two di�erent culture media: on the left, the sulfur metabolism medium; on the right, the devulcanization medium, with tire powder.

Figure 2. Left: RoxA structure. There are three loops (forming a tower) that coordinate polyisoprene cleavage, in HEME 1 (Ref. Lcp RoxA Right: Lcp bioinformatics.

Rubber degradation enzyme

Lcp (Latex clearing protein)

RoxA (Rubber oxygenase A)

Products Small polymers ODTD Molecular Weight 42kDa 72kDa

Heme group Unbounded (n-type) Two-Bounded (c-type) Structure - PDB 4B2N

OO

ENZYME DINAMICS

Once the degradation occurs in extracellular medium, it is important to have an e�cient exportation system. A new biobrick submitted by our group is detailed below.

PROMOTERS

ENZYMES EXPORTATION

Our main circuit depends on an inducible promoter. Aiming the best result for industrial application, we designed two types of circuits to test the viability of three di�erent inducible promoters in DH5α and BL21 E. coli strains: Plac, Para and Prha.

Figure 6. TAT signal secretion scheme. The peptide is cleaved and the protein is released to the extracellular medium

Figure 7. OmpA is already an iGEM part. It makes possible to anchor a protein outside of the bacteria

Figure 4. Zero means no induction. Left: BL21 with circuit 1 under pLac induction. Right: BL21 with circuit 1 under pRham induction.

TEST 1

TEST 2

INTERLAB STUDY

MODELINGGene Expression ENZYMATIC KINETICS

ENZYME PRODUCTION

KILL-SWITCH REGULATION

L = NUMBER OF BREAKABLE POINTS IN THE POLYMERS

KILL-SWITCH REGULATION

KILL SWITCH

Looking at the main circuit we have:- rhamnose , [tetR], inhibiting HokD formation, cell lives.- rhamnose , [tetR] and [HokD], cell dies.

RUBBERBYE - DEGRADING RUBBER TO FUELRUBBERBYE - DEGRADING RUBBER TO FUEL

WET LAB BEYOND THE BENCH

ACHIEVEMENTS

FUTURE REMARKS

economy to input tire waste into a value chain.

We intend to continue the circuit assembly of exportation tests, analyzing the viability of E. coli for this process. If it doesn’t work, we will use another chassis that has a naturally exportation system of proteins; In the devulcanization process, we will cultivate Acidithiobacillus ferrooxidans in a appropriate period of time to achieve the production of TetH;Finally, we aim to express roxA an Lcp solubles to verify its feasibility for an industrial process. The optimization of the kill switch will continue, in order to improve a future scale-up.

Ref. Lcp RoxA 1: Birke, Jakob, Wolf Röther, and Dieter Jendrossek. "Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b-Type Cytochrome and Di�ers from Rubber Oxygenase A (RoxA) in Its Biophysical Properties." Applied and environmental microbiology 81.11 (2015): 3793-3799

Ref. Lcp RoxA 2:.Seidel, Julian, Georg Schmitt, Maren Ho�mann, Dieter Jendrossek, and Oliver Einsle. "Structure of the processive rubber oxygenase RoxA from Xanthomonas sp." Proceedings of the National Academy of Sciences 110, no. 34 (2013): 13833-13838.

Tire accumulation is one of the main environmental problems worldwide. Rubber waste prevelant destination is incineration for generating energy, although other possibilities - such as reuse and recycling - also exist. Here, a innovative solution for better use of scrap tires ecconomically and environmentally is proposed. We developed a system to degrade the tire main composite, polyisoprene, and transforme it into high-valued products. It was designed a three-step procedure: devulcanization, degradation and fuel generation. We aim to scale these up to bioreactors .