Team:METU Turkey project

Degredo PETronum!

ABSTRACT

The usage of polyethylene terephthalate-PET (as like plastic bottles or textiles) is one of the big problems of our era. Many plastic bottles are not reusable and are not safe for hot liquids or for use in dishwashers or microwaves. Some tougher, reusable plastic bottles may cause cancer. The main disadvantage of plastic bottles is that so many of them end up in landfills instead of being recycled. It takes thousands of years for plastic bottles to biodegrade, and the degrading process emits toxic chemicals into the air. We may alter the destiny of plastic in useful way.

Our Abstract in French!

Degrado PETronum La présence de poly-téréphtalate d'éthylène (PET) dans le plastique, comme celui des bouteilles, est l’un des problèmes majeurs de notre époque. La plupart des bouteilles plastiques ne sont pas réutilisables. Et les plus robustes, réutilisables, pourraient être impliquées dans des cancers. Le principal inconvénient des bouteilles en plastique est que nombre d'entre elles finissent dans les décharges au lieu d'être recyclées. Il faut plusieurs centaines d’années pour que ces bouteilles se dégradent,et leur dégradation est à l’origine d’émission de toxine dans l’air. Grâce à ce projet, l’environnement serait débarrassé des PET tout en produisant du pyruvate qu'E.coli serait capable d’utiliser comme source de d’énergie en l’incorporant à son cycle cellulaire. Nous allons dégrader le PET en catéchol. Puis, ce catéchol résultera en pyruvate par le 2-hydroxymuconate.

OUR MISSION

Our project aims to clean enviroment with the degredation of PET to pyruvate by E.coli. With this project,while the environment will be cleaned from PET, the E.coli would add pyruvate to the cell cycle and use it as a new source. We divide our project into two parts. At the first part we found gene design for converting the PET into 2-hydroxymuconate semialdehyde in iGEM database. The arrangement at the bottom explains the first part:

At the second part, we wanted to go further and researched the gene parts for convertion of 2-hydroxymuconate semialdehyde into pyruvate. This part will give us the chance of adding new parts to iGEM database. In this arrangement, it can be observed in detail:

Why do we want to degrade PET? As you know, plastic smears into the soil and water easily, although plastic producing companies claim that, they collect the plastic lower quality available again and this way allows recycling , it is possible to see PET in water and soil. Our aim is to separate freshwater(rivers and lakes), salt water(sea and oceans) and the most important, the soil from this carcinogen with biodegradation method which we design with E.coli. Despite of recycling of PET, after a while it would be a substance which is unusable and unrecyclable and because of this reason we want to design an organism to degrade it.

How the PET smears to the lakes? Ultimately, people live close to water resources, at the same time betray water. Plastic smears water at different forms( like bottle,bag or pot) and the damage increases with uncollected years. Derraik, J.B, (September 2010). The pollution of the marine environment by plastic debris: a review. Marine Pollution Bulletin, 44(9), 842-852. Retrieved from http://www.sciencedirect.com/science/article/pii/S0025326X02002205

How does our project work?

Totally degradation of PET to pyruvate is done by E. coli.

As an analog: The project depends on E. coli that eats PET as we eat sandwiches.

The Circuit of our Project

The Circuit of our Kill Switch Model

We made a design for collected plastic and aim to having a bacteria pool of plastic which our organism degredate PET without producing toxic material. In this design, controlled biodegradation is very important because we can not precise all the risks. So, we made our organism addicted, briefly it works with a kill-switch mechanism with the presence of arabinose.

Why did we choose this project?

The biodegradation of plastic bottles took thousands of years, and the degrading process emits toxic chemicals into the air. Our project aims to clean enviroment with the degredation of PET to pyruvate by E.coli. With this project,while the environment will be cleaned from PET, the E.coli would add pyruvate to the cell cycle and use it as a new source.

Can our bacteria degrade PET in huge amounts?

For nowadays, yes. Also we can do this biodegradation process with different organisms like algeas in water. For soil, situation is more complicated but actually, we try to obtain this biodegradation genes from a soil organism Pseudomonas putita. E.coli can be worked as easy as P. putida and has a good adaptiton. If we talk about a biodegradation on a collected plastic, our project would work properly.

Why did we associated some of our parts with Pseudomonas putida F1 strain?

P. putida is used for related experiments and it has lots of metabolic similarities with E.coli

Could we use P. putida F1?

Unfortunately, it is easy to handle but obtaining a gene is very tough. The errors in the experiments show us there is a problem in the primers. Because of limited budget and time, we could not obtain F1 gene, but constructed our project in E. coli DH5a.

Why we prefer to obtain pyruvate as end product, instead of Acetyl-CoA?

Because we made some regulations anchancements, and additions on what 2010 Toronto iGEM team did by ending up with Acetyl-CoA. Here you can see its details https://2010.igem.org/Team:Toronto/Project

Which teams worked on the previous parts of our project?

With the experiments of Darmstadt, London Imperial college and UC_Davis Teams and other iGEM parts, we can obtain catechol from PET. For going further, we ligated K316003 part with a constitutive promoter. So, we reduced huge amounts of catechol into 2-hydroxymuconate Semialdehyde and obtained green colour as we expected. Then, we designed a new part From https://2012.igem.org/Team:TU_Darmstadt/Parts BBa_K808000,BBa_K808010,BBa_K808011,BBa_K808012,BBa_K808013,BBa_K808014 From http://parts.igem.org/Part:BBa_K316003 and Bba_K316003 http://parts.igem.org/Part:BBa_K936020 and Bba_K936020 With these parts, we can obtain catechol from PET. Our project starts after catechol and we aim reduce catechol to pyruvate to add cell metabolism!

Our Future Aspect:

In our early rough drafts’ of our project we focused on PET degredation until Catechol but when we obtained that Catechol can not use in biological pathways as biproduct, we decided to focus on PET degredation until pyruvate which is a substance that can be used by organisms directly in their metabolic pathways. We knew from our researchs of past iGEM projects, there are some great projects about generating electric from E. coli and some other microorganisms by MFC. MFC (Microbial Fuel Cell) can be utilized for electric generation through the conversion of organic and inorganic substrates by microorganisms. We read wiki sites of some iGEM teams ,especially Bielefeld 2013 and TU Darmstadt, which was about generating electricity through MFC. We know that through the reactions of PET degradation until pyruvate, after pyruvate metabolism there is overproduction of H ions and we think that excessive H ions can generate potential difference which can be further used for electric generation. Can you imagine we can live in such a world, while generating electricity it destroys all plastic wastes!