Sporulation Knockout

One of the main aims of our project was to knock out sporulation in B. subtilis to provide a longer lasting biofilm. We aimed to achieve this by knocking out the spoIIE gene in B. subtilis, which encodes a phosphatase that plays a major role in spore formation.

To test if the ΔspoIIE strain successfully knocked out sporulation, we performed a spore count assay, where cells are cultured in starvation media to induce sporulation, followed by heating some of the samples at 70 °C to kill any cells that are not spores. The number of spores and the number of vegetative cells can then be determined by serially diluting and plating on LB agar to count the colony forming units (CFUs).

The below figure shows the number of vegetative cells and spores for wild type B. subtilis 3610 and ΔspoIIE B. subtilis 3610 – these results show almost a total loss of spore formation in the ΔspoIIE strain: around 1 in 4 of the wild type culture are spores, whereas only 1 in 20,000 of ΔspoIIE culture are spores!

As the goal of knocking out sporulation was to increase the long-term stability of the biofilm, we also wanted to check how the biofilm biomass of ΔspoIIE compared to wild type B. subtilis over time. For this, we optimised a biofilm biomass assay by staining with safranin – as seen below, there does not appear to be significant amounts of biofilm dispersal/breakdown in wild type by 2 weeks (likely due to these assays being carried out in TSB, a nutrient rich media), so from this data we cannot conclude if ΔspoIIE is less affected by biofilm dispersal; however, ΔspoIIE appears to form a mature biofilm significantly faster than wild type B. subtilis and appears to have slightly greater total biofilm biomass overall, hence also achieving our aim of increased biofilm formation

Increased Biofilm Formation

To achieve increased biofilm formation, in addition to ΔspoIIE, we designed constructs for inducible expression of multiple regulatory components of the B. subtilis biofilm formation pathway, in addition to inducible expression of the major protein component of the biofilm matrix.

We tested the biofilm formation of these engineered strains in the presence of the inducer, xylose, using the same safranin stain biofilm biomass assay. These results showed that overexpression of the genes sinI, the tasA> operon and mstX all result in significantly increased biofilm formation rate, compared to wild type B. subtilis.

Decreased Pathogenic Biofilm Formation

We then wanted to confirm that protective biofilms can indeed reduce the incidence of surface attached pathogenic or corrosive microbes. To do so, protective B. subtilis biofilms were first formed in wells of 96-well plates, followed by introduction of a model pathogen – a strong biofilm-forming strain of non pathogenic E. coli containing an ampicillin resistance plasmid – into wells with or without these protective biofilms; after 24 hours, the number of surface-attached cells in wells with or without the protective biofilms were determined by washing, scraping the surface of the wells, and CFU counting on ampicillin plates.

The below results show that the protective biofilms significantly reduced the incidence of pathogenic biofilms, by around 100,000 fold. Additionally, our data shows that B.Max appears to be even more effective at preventing pathogenic biofilms than wild type B. subtilis.

Biofilm Formation on Common Farm Surfaces

Whilst all of our previous experiments were performed in polystyrene plates, it is important that the protective biofilms can also form on surfaces commonly found in farms. Based on our meeting with Dr Briandet, we learned that common materials used in agriculture include stainless steel and PVC. Therefore, we 3D printed plastic wells which were adhered to the surfaces of stainless steel and PVC sheets; B. subtilis cultures were then introduced into the wells as in previous assays, and after 72 hours of incubation the the media was removed, the wells were washed, and biofilm formation was identified visually.

These assays show that B. subtilis can indeed form biofilms on both stainless steel and PVC (stainless steel on left, PVC on right). Hence, B. subtilis seems an appropriate choice for applications of protective biofilms in agriculture.

Confocal Imaging of Biofilms

Additionally, during our meeting with Dr Briandet, he advised that confocal microscopy could be a valuable technique for visualising the ultrastructure of the biofilm. Whilst his lab and seemingly most papers in the literature utilise fluorescent stains such as Syto 9, these are fairly expensive and not available to many iGEM teams. Therefore, we tested confocal imaging utilising a strain of B. subtilis 3610 expressing a red fluorescent protein (mScarlet, kindly provided by Dr Diana Fusco). We found that this was effective for viewing mature biofilms grown in glass bottom microscopy dishes, as shown below by the 3D image of a mature B. subtilis biofilm captured by confocal microscopy.

Whilst we hoped to utilise confocal microscopy to investigate the biofilm ultrastructure of our engineered strains, we were unfortunately unable to do so in the time we had available.

Biosensor

We successfully assembled and transformed an AI-3 sensing construct into E. coli DH5α, however since DH5α likely does not produce AI-3, the transformant did not produce any detectable fluorescence. We later changed our focus to identifying if QseC can be expressed and localised to the plasma membrane of B. subtilis. We created a QseC-sfGFP fusion protein and confirmed the size of the construct with gel electrophoresis, however we unfortunately did not have enough time to transform it into B. subtilis to visualise the localisation.