Team:Paris Bettencourt/Biomaterials

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@iGEM_Paris

BIOMATERIAL

PHACalcium CarbonatePolysilicate

PHA

Why P3HB?

Poly-3-HydroxyButyrate (P3HB) is the perfect biomaterial to demonstrate our 3D control. It is a bioplastic already used for 3D printing. However we produced our P3HB with our own E.Coli DH5 alpha strain using the BBa_K1149051 biobrick (Imperial College London 2013) from the iGEM registry. After successfully cloning it into our bacteria and characterising the production with flow cytometry, we modified the biobrick by adding a cell-lysis system.

What is P3HB?

P3HB comes from the large family of polymers called polyhydroxyalkanoate (PHA). We were interested in using this biomaterial not only for its mechanical properties, but also for its ecological effects as it is a biodegradable plastic.
In nature, microorganism such as Ralstonia Eutrophus produce P3HB in response to physiological stress. It is used as an energy storage ready to be metabolized when they are in dire nutrient conditions.

The gene comes from Ralstonia Eutrophus H16, a gram-negative bacterium producing P3HB with a 3 enzymes pathway: PhaC, PhaA and PhaB. The first enzyme PhaA codes for 3-ketothiolase, its role is to combine 2 molecules of Acetyl-Coa into Acetoacetyl-Coa. Then it is reduced by Acetylacetyl-Coa reductase, coded by PhaB, into (R) - 3 - Hydroxybutyryl-Coa. At last, P(3HB) synthase, coded by PhaC, polymerise the latter product to form Poly-3-Hydroxybutyrate or P3HB.

Confirmation and characterization

We stained our cells using a Nile Red solution (0.3mg/mL in DMSO). Nile red is a lipophilic stain that can be used to detect P3HB presence due to red fluorescence. Thus, to characterize the production of P3HB, we used Fluorescence-activated cell sorting (FACS) a type of flow cytometer, specifically the FL2 (575 BP filter) and FL3 (620 BP filter) channels to measure the intensity of the fluorescence of the Nile Red (excitation wavelength between 520 and 550 nm, and emission wavelength between 590 and 630 nm) stained cell containing P3HB.
We used Flow Cytometry to characterize the part as we believe it is the best technique compared to Gas Chromatography/ Mass Spectrometry. Using fluorescence-activated cell sorting permit us to do hundreds of samples a day at minimal price whereas using GC/MS is not only expensive, but you can only run a few samples a day.

Cell-lysis

To link our P3HB production to our project, we needed a way to extract the product without using any chemicals or tampering with the cells. Implementing a cell-lysis system into the bacteria enabled us not only that, but also to fulfill our safety concerns.

By shining lights on our cells producing P3HB, the cell-lysis system is activated, meaning it breaks down the bacteria, therefore releasing the product out of the cell. The P3HB will then form an aggregate with the other P3HB granules around it. By orientating the lasers to specific positions, the P3HB keeps on aggregating until we have the final product.

Application

P3HB as a range of application frommedicalto bio packaging bio packaging. As it is biodegradable and renewable when composted, P3HB gets a lot of attention, and for the right reasons. Many new companies are now producing the thermoplastic, such with a production capacity of over 10,000 tons per year.
Therefore we believe P3HB and PHAs in general will be a material of the future. This is one of the reason why we chose to use this biomaterial for our proof-of-concept, on top of its physical properties that would allow the consumer to use our P3HB as a regular material for 3D printing.