Product Safety

Biocontainment: Overview

Whilst B. subtilis is generally recognised as safe and is already used as a probiotic for humans, plants, and animals, it is important that our engineered strain does not end up where it was not designed to be and possibly cause unintended consequences. Additionally, although our engineered strain is designed to minimise the probability of invasion, over time there is the possibility of these beneficial biofilms being invaded by pathogenic or corrosive microorganisms, which may then be difficult to remove during cleaning. Hence, we recognised the need for biocontainment to prevent unintended spread of B.Max, in addition to the need for inducible biofilm disruption (self-destruction) to allow for effective cleaning of any invading microbes (see Engineering).

Biocontainment - Preventing Unintended Spread

To prevent the unintended spread of B.Max, we considered 3 of the major biocontainment mechanisms – a toxin-based kill switch that results in toxin expression and cell death under certain condtions, engineered auxotrophy that only allows cells to survive in the presence of supplemented nutrients, or conditional expression of essential genes under certain conditions. As B.Max may be broadly applied over large surfaces with varying nutrient compositions, engineered auxotrophy did not appear to be reasonable in this case. Additionally, when considering the long-term evolutionary stability of the biocontainment mechanism (i.e. how easy it is to break it), toxin-based kill switches appear significantly less evolutionarily stable (i.e. easier to break) than conditional essential genes. Hence, we decided to utilise conditional essential genes for this biocontainment mechanism.

As B.Max is intended to survive only in the biofilm-associated state, we decided to design a biocontainment mechanism that allows biofilm-associated cells to survive, but results in the death of planktonic cells. To do so, we considered utilising the biofilm master regulator in B. subtilis, SinR – SinR binds to an operator region upstream of the promoter of genes involved in biofilm formation, repressing expression of these genes during the planktonic state. During the planktonic-to-biofilm transition, SinR is sequestered by SinI, preventing binding of SinR to the operator regions and allowing for expression of biofilm-associated genes. Hence, adding a SinR-binding operator region upstream of the promoter of an essential gene may allow for the desired biocontainment mechanism of survival in biofilm-assocaited state and death in planktonic state.

However, a single SinR-binding operator region upstream of the promoter of a single essential gene also appears evolutionarily unstable (i.e. it could be easily broken by disruption of the SinR-binding operator region). To increase the evolutionary stability of this biocontainment mechanism, we considered adding multiple binding sites for SinR, and binding sites for other transcription factors involved in biofilm formation, upstream of the promoters of multiple essential genes. From this, the probability of all binding sites being disrupted for all of the conditional essential genes would appear to be very low.

As B. subtilis would be planktonic during culturing and application, in this biocontainment system the cells would die prior to being able to form a biofilm. Hence, we considered inserting an additional copy of the essential gene at a different site in the genome, expressed under an inducible promoter. This would effectively form an AND gate for survival, where cells have to either be in the presence of an inducer (which could be provided during culturing) or in the biofilm-associated state.

To identify the optimal essential genes to conditionally express, we analysed the BsubCyc database for every essential gene in B. subtilis to identify those which have annotated promoter regions supported by experimental evidence and contain only a single gene under the control of that promoter (to avoid unintentionally making other genes conditional). From this, we identified gyrA, gltX, and rpsD as promising candidates.

For designing the operator region, we decided to utilise the naturally-occuring operator regions upstream of the eps and tapA-sipW-tasA operons (encoding the major polysaccharide and protein components of the biofilm matrix, respectively). These operator regions are well-documented based on experimental evidence, hence it was possible to design constructs to insert these operator regions upstream of the promoter of the essential genes, with the same distance between the operator elements and transcription start site that occurs in the eps and tapA-sipW-tasA operons.

Biocontainment - Inducible Biofilm Disruption

To allow for effective removal of B.Max and any invading microbes during cleaning, we considered the optimal mechanisms for inducible biofilm disruption. Whilst there is data supporting biofilm disruption by application of the DNase, DNase I, and the protease, proteinase K, these are eukaryotic proteins which are unlikely to function optimally in B. subtilis. Instead, we considered other proteins, such as the bacterial DNase, NucB, the bacterial proteases, NprB and Bpr, and the bacterial racemase, YlmE. Expression of some combination of these proteins following application of an inducer may result in significant amounts of biofilm disruption to allow for effective cleaning.

Safety during Outreach

Safety during Biomakespace

While we wanted to show the participants at the Biomakespace meeting the sporulation knockout strains we produced, we had to ensure that the microscope slides were properly sealed and safely transferred to the location of the meeting. Since there is no biological waste management at Makespace, nor are there proper lab spaces, we had our strain risk assessed as well.