Team:UChile-OpenBio

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Project

Overview: Background

Each year, 130 million tons of fossil plastics are produced in the world, which take 500-1000 years to degrade, and pollute the environment; 1,5 millions of marine animals were killed in 2014. A sustainable initiative is to produce biodegradable plastics; however its synthesis process (chemical and biological) is complex and expensive.

The team UChile-OpenBio is designing two populations of Bacteria to achieve this: Escherichia coli to produce a biodegradable plastic called PLA (Polylactic acid) from easy to assimilate renewable resources.

In this way, the team would help fighting against pollution, contributing to a better world!

General Concepts

A genetic module is constituted of 4 elements, called parts, that are DNA sequences which possess a specific function: the promotor, the RBS, the coding sequence and the terminator.

The role of a genetic module is to produce certain quantity of a protein under some specific conditions. The fabrication process, from DNA to protein, consists in two steps: transcription and translation.

If the DNA is a book of recipes, the transcription process consists in photocopying one page, in which are written the instructions for one specific food; and the translation process corresponds to the cooking phase, in which the "cooker" is the ribosome. The ribosome reads the instructions and at the same time builds the protein. This is called the Central Dogma of Biology.

Scheme of the general concepts

General Concepts

The transcription can be regulated by different factors (repressors, pH, light, etc) which are detected by the promotor. These signals induce or inhibit the activation of the module.

If the module is activated then the transcription process begins, in which several copies of the message are made.

The RBS (Ribosomal Bonding Site) intervenes in the translation process: if it is strong many proteins will be synthesised. If it is weak, few proteins will be synthetized. So the function of RBS is to regulate the quantity of produced proteins.

The coding sequence is the message, the recipe that shall be translated to protein.

The terminator is a DNA sequence that puts an end to the transcription process.

Why Legos are a friendly way to explain SB?

The team decided to used legos to explain the pla project due to diferent reasons.

First Reason

They make tangible microscopic things

Second Reason

They allow a strong analogy with the DNA parts called « bio-bricks»

They are standard and modular like a biobricks

Third Reason

Finally, they are a funny way to explain complex concepts... for the students...and the teachers!

Overview: Lego description

E. Coli 1 Poblation: “Lactadora”

In our project we used two bacterias « Escherichia coli »: name and first name of the bacteria.
This population are responsible for lactate production.

E. Coli 2 Poblation: “PLAdora”

« Escherichia coli »: name and first name of the bacteria. « 1 » y « 2 » only mean that these bacteria, in this process, are responsible for different functions (but they belong the same bacteria strain).
This population are responsible for process the lactate into PLA.

The « Lactate DeHydrogenase » (LDH) is responsible for lactate production.

Transformation of glucose into pyruvate is a bacterial natural process, it doesn’t need human intervention.

LDH uses pyruvate and transforms it into lactate, the intermediate compound that allows PLA synthesis.

Finally, lactate can be obtained from glucose thanks to LDH, which gene we insert into the bacteria.

According to last image, the module which produces LDH is indirectly responsible for lactate production, for it will be symbolized as if the LDH directly produced lactate.

The lactate produced by the E.coli 1 Population is secreted into the medium and enters in the bacterias of the E.coli 2 Population

Lactate enters into the E.coli 2 Population to be processed into PLA by the presented module.

The lactate convertion into PLA is driven by two enzymes: « P-CoA-T »: Propionyl-CoA-Transferase « phaC1 »: PolyHydroxy Alkanoate –Class 1

The P-CoA-T unites lactate with a cofactor called « Coenzyme A », transforming lactate into « Lactyl-CoA ».

PhaC1 gathers all the lactyl-CoA that are present in the cell and unites them together...

This reaction is called « polymerization ». The product of this polymerization is the PLA.

To sum up, this module processes lactate to transform it into PLA.

The produced PLA is processed by a second module, which produces a hybrid protein constituted of a first protein called phasyn and of a much smaller protein having affinity for PLA. The role of this hybrid protein is to make the cell know that PLA is ready for exportation.

The hybrid protein sticks with PLA and the cell exports it into the extra-cellular medium, ready to be extracted and purified.

Importan Modules of the process

E.coli 1 Population

E.coli 2 Population

Glucose transformation into lactate

Transformation of lactate into PLA

PLA exportation

Regulation of lactate production in E.coli 1

When being outside the cells, lactate acidifies the medium, which is dangerous for the bacteria. That’s why a pH-sensing module has been built to detect pH levels and produce the TetR protein when the pH is lower than 5,5.

When being outside the cells, lactate acidifies the medium, which is dangerous for the bacteria. That’s why a pH-sensing module has been built to detect pH levels and produce the TetR protein when the pH is lower than 5,5.

TetR is a repressor of the lactate production module. It means, when pH becomes lower than 5.5, the lactate production is shut off by TetR. This shrinks the lactate concentration in the medium so that the pH increases again.

Communication between E.coli 1 and E.coli 2

In fact, the module that produces lactate not only produces it but is also responsible for the synthesis of a small molecule called HSL (HomoSeryl-Lactone) . This molecule is exported into the medium and is able to enter the second bacteria, being responsible for communication between E.coli 1 and 2.

Inside the E.coli 2 bacteria, is also inserted a module which produces a protein called LuxR.

The concerted action of luxR with HSL activates the other modules of the E.coli 2 (logic door « AND »),

Communication between E.coli 1 and E.coli 2

The concerted action of LuxR with HSL activates the PLA production and makes possible its exportation. This activation is possible only if there is enough lactate in the medium (since it is the same module that produces lactate and HSL). This way, E.coli 1 leads the production, telling to E.coli 2 when the PLA synthesis can begin). That’s why it is said that HSL is a communication molecule.

The concerted action of LuxR with HSL activates the PLA production and makes possible its exportation. This activation is possible only if there is enough lactate in the medium (since it is the same module that produces lactate and HSL). This way, E.coli 1 leads the production, telling to E.coli 2 when the PLA synthesis can begin). That’s why it is said that HSL is a communication molecule.

Overview: Main goal

Main goal

For the iGEM competition, the team aims to engineer a biological system, enabling it to degrade glucose in order to produce and export into the medium a biodegradable plastic called PLA.

Goal 1

Designing and implementing a self-regulated lactate production system which will allow light green bacteria (Figure 1) to control the lactate production by pH-sensing: the higher lactate concentration, the lower the pH, which induces a negative control in the first population of E.coli, stopping the production of lactate and by the way, of PLA.

Goal 2

Designing and implementing a PLA production and exportation system which will allow blue bacteria to send the biological PLA outside the cells, into the medium. This way, the purification of the bioplastic would be easier.

Goal 3

Designing and implementing a safety system, which will consists in making arabinose-dependent the cell survival. If the medium contains arabinose, bacteria will grow up, but if bacteria escape from their medium, the cells will produce a toxin which will kill them. This way, we will ensure the safety of the persons working in the laboratory and of the environment.

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