Team:Paris Bettencourt/Biomaterials

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, microorganisms such as Ralstonia Eutrophus produce P3HB in response to physiological stress. It is used as an energy storage ready to be metabolised when nutrients become scarce.
The gene comes from Ralstonia Eutrophus H16, a gram-negative bacterium producing P3HB thanks to 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. The newly formed Acetoacetyl-Coa is reduced by Acetylacetyl-Coa reductase, coded by PhaB, into (R) - 3 - Hydroxybutyryl-Coa. At last, P(3HB) synthase, coded by PhaC, polymerises 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), 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 allowed 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.
Flow cytometer analysis of cell stained with NileRed with BBa_K1149051

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 has a range of application from medical to 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, so much that it reaches 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 reasons 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.
CALCIUM CARBONATE

Introduction

In recent years, the interest in obtaining microbial cement has gained popularity. This is in part because of the potential of microbial cement to overcome problems such as fractures and fissures in concrete structures which are created by weathering, land subsidence, faults, earthquakes and human activities. Synthetic biology has proposed a novel way to repair and remediate these problems. One of the possible solutions 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 revolutionise them.

Figure 1: Chemical structure of calcium carbonate.

Back to basics

Biomineralization is process by which living organisms are naturally able to produce minerals. Production of microbial calcium carbonate (CaCO3) is a widely studied and a promising technology with various engineering applications. The use of CaCo3 include: treatment of concrete, manufacturing of construction materials (such as building bricks and fillers for rubber), synthesis of plastics and inks.
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.

Figure 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.
Figure 3: Stained calcium carbonate deposits formed in the present of CARPs in artificial seawater(ASW).

The most commonly found mechanism in bacteria for CaCO3 precipitation has been to generate an alkaline environment through different physiological actions. Precipitation of CaCO3 by ureolytic bacteria is the most straightforward and most easily controlled mechanism of microbially induced calcium carbonate precipitation. It also has the potential to produce high amounts of carbonates in short period of time.

Alternative

Besides the CaCO3 precipitation induced naturally by 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).

Figure 4:The pathway of calcium carbonate precipitation through production of coral acid-rich proteins in E.Coli.

In our project, coral acid-rich proteins (CARPs) was cloned and expressed in E.coli BL21 strain. They were characterized for their ability to induce calcium carbonate precipitation.

According to the putative mechanism of calcium carbonate nucleation by CARP, a highly acidic pocket brings together a calcium ion and a carboxylate molecule thus favouring their reaction (Figure 5). Evidence based on high-resolution magnetic resonance spectroscopy has shown that the calcification in stony coral is mainly controlled by CARPS embedded in skeleton organic matrix.

Figure 5: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, favouring 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 key advantage of CARPs is their power to bypass the acidification of the growth medium and the urea synthesis associated with the classical urease pathway. Furthermore, CaCO3 precipitation with CARPs occurs in one enzymatic step, greatly reducing the metabolic cost for the cell.

Figure 6: SDS-PAGE separated CARP1-CARP4 proteins according to their molecular weight, based on their differential rates of migration through a sieving matrix (a gel) under the influence of an applied electrical field.