Team:UChile-OpenBio/Description
Titulo
Titulo
Titulo
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.
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.