Difference between revisions of "Team:Paris Bettencourt/Biomaterials"

Revision as of 17:39, 1 November 2017

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.

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.

Introduction

In recent years, the interest in obtaining microbial cement has gained its popularity along with such problems as fractures and fissures in concrete structures which is created by weathering, land subsidence, faults, earthquakes and human activities. Synthetic biology has proposed a novel way to repair and remediate problems. One of the possible solution is biomineralization of calcium carbonate using microbes such as Bacillus species. The application of microbial concrete in construction may simplify some of the existing construction processes and revolutionize them.

Fig. 1: Chemical structure of calcium carbonate.

Back to basics

Biomineralization is a natural process in living organism by which they are able to produce minerals. Production of microbial calcium carbonate (CaCO3) is a widely studied and a promising technology with many uses in various engineering applications, for example: treatment of concrete,construction materials such as building bricks and as fillers for rubber, plastics and ink. There are three distinct pathways of bacterial calcium carbonate precipitation:

1) biologically controlled - cellular specific control of formation of the mineral (exoskeleton, bone or teeth),

2) biologically - influenced - passive mineral precipitation caused through the presence on the surface of the cell of organic matter and

3) biologically- induced - which is the chemical alteration of an environment by biological activity.

Fig. 2: Alizarin Red staining for detection of calcium carbonate composites in the precipitated proteins: A) stained calcium carbonate powder - positive control, B) stained sample of BL21 extracted protein precipitation in CaCl 1M solution - negative control , C) stained sample of CARPs extracted protein precipitation in CaCl 1M solution.

The most commonly found mechanism in bacteria for calcite precipitation has been to generate an alkaline environment through different physiological actions. Precipitation of carbonates via urea hydrolysis by ureolytic bacteria is the most straightforward and most easily controlled mechanism of microbially induced calcium carbonate precipitation with the potential to produce high amounts of carbonates in short period of time.

Alternative

Instead of calcite precipitation from natural microbes, many other organisms also have the power to produce calcium carbonate, such as corals. In the stony coral, Stylophora pistillata, 4 acid-rich proteins (CARPs 1–4; GenBank accession numbers KC148537–KC148539 and KC493647) were identified to be responsible for calcium carbonate precipitation. These proteins were found in the study of changes in the growth of corals with increasing of acidity in the ocean.
As such, bioreaction of calcite formation is far from the thermodynamic equilibrium. It may even compromise with acidification and very low mineral saturation state (E. Tambutté & A. A. Venn et al. 2015).

Fig. 3: The pathway of calcium carbonate precipitation through production of coral acid-rich proteins in E.Coli.

In our project all coral acid-rich proteins (CARPs) was cloned and expressed in E.coli BL21 strain and characterized for ability of calcium carbonate precipitation.

The putative mechanism of calcium carbonate nucleation is that highly acidic pockets of CARPs localize with the substrate and buffer thus catalyzing the reaction between calcium ion and carboxylate (Fig. 2). Through high-resolution magnetic resonance spectroscopy analysis, evidence have been shown that the calcification in stony coral is mainly biologically controlled and relatively robust, due to template-induced nucleation mediated by the skeleton organic matrix, in particular, acid-rich proteins like CARPs.

Fig. 4: The highly acidic regions of the proteins interact with calcium ions (grey spheres) via coordination chemistry allowing the carboxylate groups to attract and localize calcium ions in a microenvironment, enhancing the local ionic strength. This local interaction results in a shift in pKa, favoring the formation of carbonate. Being a stronger Lewis base, with greater negative charge, carbonates displace carboxyl groups from the proteins to form stable coordination bonds with the calcium on the protein scaffold.

The positive aspect of this method is that we can increase the production of microbial calcium carbonate by reducing its sensitivity to changes in the level of acidity in the environment, as well as get rid of side products in our system such as urea. Furthermore, this is a one enzyme pathway, allowing the cost of the cells to be greatly reduced.

Silicate (Si_mO_n)is one of the known form of biomineralization, the main component of planet’s crust, and of many synthetic materials. It is used a lot for electronic and biologic microimplant. The physical properties depend entirely on how the silica crystals are organized, quartz, glass or others. Mineral polysilicate is formed with a great pressure and temperature. It is the variation of those two factors that induce the formation of different kind of rocks. Some living organism took advantage of the abundance of silicate in there environment and used it to create there skeleton or there shell. Some sponges that can grow up to 3m have skeleton made of polysilicate. Diatoms, unicellular microalgea, can also can also cover there cell wall in silica. The formation processes in sponge and diatom are fairly well known. The pathways require multiple proteins, but the key factors have been successfully expressed in E.coli.

We decided to use Silicatein α from the sponge Suberites domuncula because it has already been used in iGEM before. First we used the biobrick Bba_K1890000 from the 2016 TU Delft team, that they kindly agreed to send to us. We created a construct in PsB4K5 (http://parts.igem.org/Part:pSB4K5) using Pbad (http://parts.igem.org/Part:BBa_K206000) as a promoter and p0015 (http://parts.igem.org/Part:Bba_B0015). After the production culture, we stained the cells with rhodamine 123 to test the presence of poysilicate. As you can see on the graph, the three populations supposed to produce silicateinα doesn’t show any fluorescence that would indicate the presence of polysilicate.

After that, we used a biobrick from iGEM Pasteur, that was designed a previous year but never used nor submitted. This biobrick was designed by replacing the active site of the cellulose producing gene by the active site of Silicateinα. We obtain some pretty good result with it. The facs results we obtain from those cell clearly show a different population between the stained control and the three cell line with

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France

bettencourt.igem2017@gmail.com

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