Protective Biofilm
With B.Max, we have developed the basis for engineering an effective protective biofilm – whilst we have focused on the applications in agriculture and corrosion, this concept could also be applied to many other fields, such as preventing disease or reducing pesticide use in crops, or even in the medical setting. The concepts we have established in B. subtilis may also be useful for engineering protective biofilms utilising different species.
Additionally, the parts we have designed may also be useful for teams working with B. subtilis outside of protective biofilms – such as the bacterial nuclease, proteases, and racemase for biofilm degradation. These parts may be useful in future projects working on biofilm degradation, such as projects focused on the major issue of pathogenic biofilms in medicine preventing effective treatment.
The parts we have developed for increasing the rate of biofilm formation, increasing total biofilm biomass, sporulation knockout, and biofilm degradation are available in our Parts Collection.
Integration Sequences
As part of our project, we utilised the amyE, lacA, and sacA integration sites for insertion of constructs within the B. subtilis genome by homologous recombination. These sites are commonly used within the literature for cloning into B. subtilis, and using different integration sites for different constructs allows for multiple constructs to be stably inserted into the genome of the same strain, without having to re-do the cloning for each part. We have designed and tested sequences for integration into these sites and have added these sequences to the registry for future teams working with B. subtilis to use.
B. subtilis Specific Assays
To test the effect of our constructs on biofilm formation, we needed to utilise an assay for measuring biofilm biomass. The standard assay for this is a crystal violet assay in a 96-well plate, however we found the standard protocol for this assay to be inconsistent and unreliable. Instead, we optimised a similar 96-well plate biofilm staining protocol utilising safranin as a stain – we tested for the optimal concentration of the safranin, whether the safranin was dissolved in water or water/ethanol, the solvent used for washing the biofilm, the number of wash steps, the length of the drying steps, and the solvent used for resuspending the stain. The optimised protocol is available on our Protocols page for future teams working on biofilms to have a cheap, reliable, and minimally variable biofilm biomass assay.
We also developed and optimised protocols for other assays, such as a biofilm pathogen invasion assay and a biofilm corrosion assay.
Orthogonal Nutrient Source Utilisation
Additionally, we designed constructs with the expected outcome of providing our B. subtilis strain with the ability to utilise porphyran – a polysaccharide found in seaweed, which is rarely digestible by non-marine organisms. Whilst we unfortunately did not have time to clone the constructs out of the Phocaeicola plebeius strain from which they are located, we have added the designed constructs to the registry for future teams to hopefully build upon and test.