In order to implement a Bacillus subtilis biofilm to render micropollutants in wastewater less toxic, certain requirements have to be fulfilled. First of all, the enzymes we are using have to be exported into the extracellular matrix to protect our bacteria from the toxicity of the substances. We also have to avoid those enzymes from getting washed away by the water to make sure they can convert the micropollutants. Furthermore, our biofilm as a whole has to absorb the substances to enable their enzymatic degradation. Since we have to prevent GMO release from the biofilm into the environment our biofilm needs to resist mechanical pressure to which it is exposed in wastewater treatment plants.

The Major Biofilm Matrix Component (TasA)

The Bacillus subtilis biofilm matrix consists mainly of exopolysaccharides (EPS) and proteins. One protein essential for the structure and formation of a biofilm is the major biofilm matrix component (TasA).^[1] It polymerizes into long amyloid-like fibres which are attached to the cell wall by the TasA anchoring protein (TapA) outside of the cell.^[2] The knockout of tasA leads to a mutant that forms a weak biofilm of decreased thickness.^[3] B. subtilis secretes proteins in order to enable intercellular linkages and communication and therefore uses the Sec-dependent signal recognition particle (Sec-SRP) pathway. For proteins to be secreted via the SPR pathway, it is necessary that the proteins possess a 27 amino acids long N-terminal signal peptide, which is cleaved off during the membrane translocation. Following this scheme, TasA also possesses this N-terminal signaling peptide.^[4,5] The Sec-SRP pathway consists of four main steps: 1) the signal peptide is recognized by a ribonucleoprotein complex, the signal recognition particle (SRP). 2) SRP targets the Sec translocase which transports the protein through the membrane. 3) during membrane translocation the signal peptide is cleaved off by the SipW peptidase leading to the release of the protein. 4) chaperones mediate the correct folding of the protein outside the cell.^[5]

A Fusion Protein of TasA with our Degradation Enzymes

We want to immobilize our degradation enzymes in the biofilm matrix, due to the improved stability and improved enzyme activity of immobilized enzymes.^[6] Furthermore, within the biofilm matrix the degradation targets are better accessible for enzymatic degradation than within the cytoplasm. Therefore, we decided to generate fusion proteins with our degradation enzymes and the protein TasA, following previous work by Huang et al..^[7]

They showed that fusion proteins consisting of TasA and a second protein domain (e.g. mCherry, SpyTag) are successfully exported into the biofilm matrix. Even a fusion protein of TasA with the larger enzyme MHETase (63.1 kDa) was localized in the biofilm matrix of B. subtilis. Based on the research of Huang et al., we planned the fusion protein of superfolder green fluorescent protein (sfGFP) with TasA as a proof of concept. The sequence of the sfGFP gene is codon optimized for B. subtilis and fused to the C-terminus of the tasA gene with a gene fragment encoding a glycine-serin-rich linker (ARGGGGSGGGGS). The display of the TasA-sfGFP fusion protein in the biofilm matrix can be verified using fluorescence microscopy. If TasA-sfGFP is successfully expressed in the biofilm matrix, we hypothesize that analogously designed TasA fusion proteins with our targeted degradation enzymes CotA, CueO and EreB will likely succeed.

Integration of the TasA Fusion Proteins in the B. subtilis Genome

We want to ensure that only TasA fusion proteins, but no endogenous TasA, are secreted into the matrix in order to increase the number of immobilized degradation enzymes in the biofilm matrix. Therefore, we use a B. subtilis TasA knockout strain supplied by the group of Prof. Stülke (Georg-August-Universität Göttingen).^[8] Then as a proof of concept, the TasA fusion protein will be introduced in B. subtilis via plasmids (see Huang et al.). In the next step we want to integrate the TasA fusion proteins into the genome of our B. subtilis strain. For further information on the workflow and analysis of our concept, please refer to our next on Engineering Success.

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A Atomic Force Microscopy (AFM) is a probe microscope with an up to 1000-fold higher resolution than common microscopes.^{[17] [18]} By measuring the surface, it will give precise information about the topology of our biofilm. A typical AFM setup is shown in Figure 1. The imaging process works with the offset of the laser pointing at the cantilver’s back, as well as the mechanical force bending the cantilever back.
AFM is capable of different measuring modes, that are favourable in varying conditions. For a biofilm’s soft surface, the non-contact mode is preferable. Since it reduces the friction of the cantilever’s tip on the surface.^[19]
Using our 3D printed flow chamber experiments in combination with AFM data analysis, we can determine the stability of our biofilm. How we will use the flow Chamber in Combination with the AFM you can see here.

We want to produce our pollutant-degrading enzymes fused to one of the B. subtilis biofilm-forming proteins, the major protein component (TasA). This way it will be displayed in the matrix of the biofilm. We need to make sure that the substances are able to enter the biofilm to be converted by our displayed enzymes. Here we focused on the sorption of diclofenac because it poses the biggest issue in wastewater treatment plants. Torresi et al. recently established an assay to measure the uptake of small molecules into biofilms of various thickness on which our assay is based on.^[20]

We grow the biofilm directly on carriers used in wastewater treatment to make the experiment as realistic as possible. After the biofilm is formed on the carriers, we test the diclofenac uptake. Therefore, we incubate the carriers with different concentrations of diclofenac and take samples of both the solution and the biofilm at certain time points. The biofilm sample is resuspended in water, centrifuged and washed repeatedly. After that, the cells are lysed via sonification and the suspension is centrifuged again to clear the lysate. The supernatants of this step and the samples of the diclofenac solutions are quantified via UV after HPLC separation. If diclofenac is absorbed by the biofilm at the assayed concentrations, we will do the same with concentrations that can be found in wastewater in Germany and then analyze the taken samples via LC-MS because it is more sensitive than HPLC with UV detection.^[21]

Importantly, plastics has shown adsorption of hydrophobic substances.^[22] On that account, we perform the same assay with an empty carrier in diclofenac solution to see potential adsorption to the carrier itself.