Reeksting, B. J. | Search bioRxiv

bioRxiv2022

Chimeric MerR-Family Regulators and Logic Elements for the Design of Metal Sensitive Genetic Circuits in Bacillus subtilis

Whole-cell biosensors are emerging as promising tools for monitoring environmental pollutants such as heavy metals. These sensors constitute a genetic circuit comprising a sensing module and an output module, such that a detectable signal is produced in the presence of the desired analyte. The MerR family of metal-responsive regulators offers great potential for the construction of metal sensing circuits, due to their high sensitivity, tight transcription control and large diversity in metal-specificity. However, the sensing diversity is broadest in Gram-negative systems, while chassis organisms are often selected from Gram-positive species, particularly sporulating bacilli. This can be problematic, because Gram-negative biological parts, such as promoters, are frequently observed to be non-functional in Gram-positive hosts. Herein, we combined construction of synthetic genetic circuits and chimeric MerR regulators, supported by structure-guided design, to generate metal-sensitive biosensor modules that are functional in the biotechnological work-horse species Bacillus subtilis. These chimeras consist of a constant Gram-positive derived DNA-binding domain fused to variable metal binding domains of Gram-negative origins. To improve the specificity of the whole-cell biosensor, we developed a modular AND gate logic system based on the B. subtilis natively split {sigma}-factor, SigO-RsoA, designed to maximise future use for synthetic biology applications in B. subtilis. This work provides insights into the use of modular regulators, such as the MerR family, in the design of synthetic circuits for the detection of heavy metals, with potential wider applicability of the approach to other systems and genetic backgrounds.

synthetic biology↗

bioRxiv2019

In-depth profiling of calcite precipitation by environmental bacteria reveals fundamental mechanistic differences with relevance to application

Microbial-induced calcite precipitation (MICP) has not only helped to shape our planets geological features, but is also a promising technology to address environmental concerns in civil engineering applications. However, limited understanding of the biomineralization capacity of environmental bacteria impedes application. We therefore surveyed the environment for different mechanisms of precipitation across bacteria. The most fundamental difference was ureolytic ability, where urease-positive bacteria caused rapid, widespread increases in pH, while non-ureolytic strains produced such changes slowly and locally. These pH shifts correlated well with patterns of precipitation on solid media. Strikingly, while both mechanisms led to high levels of precipitation, we observed clear differences in the precipitate. Ureolytic bacteria produced homogenous, inorganic fine crystals, whereas the crystals of non-ureolytic strains were larger with a mixed organic/inorganic composition. When representative strains were tested in application for crack healing in cement mortars, non-ureolytic bacteria gave robust results, while ureolytic strains showed more variation. This may be explained by our observation that urease activity varied between growth conditions, or by the different nature and therefore material performance of the precipitate. Our results shed light on the breadth of biomineralization activity among environmental bacteria, an important step towards the rational design of bacteria-based engineering solutions.

microbiology↗