Dervyn, E. | bioRxiv Science

bioRxiv2025

ModuloStat: An Internet of Things' Path to Continuous Cultures in Mini-Bioreactors

In continuous culture, a population of microorganisms is propagated in a stable environment over many generations. This is particularly relevant for experimental evolution and metabolic studies. However, continuous culture protocols are difficult to implement, so they are not commonly used in microbiology laboratories. Here, we present the ModuloStat, a modular, open-source framework that facilitates continuous culture in mini-bioreactors. The ModuloStat system is grounded on digital fabrication tools easily accessible in FabLabs and programmable electronics. Maintaining a culture is divided into tasks assigned to dedicated printed circuit boards with a microcontroller connected to a Wi-Fi network. According to Internet of Things principles, each board operates a set of sensors and actuators autonomously and can receive and send information. The boards are stacked to implement complex behaviors and can be modified to accommodate new features. A thermoregulated box holds the components and can be placed on a laboratory bench or transported under a sterile hood for inoculation. Sterility is ensured by autoclaving, after assembly, all components that will come into contact with the culture medium. In-situ optical density monitoring combined with modularity and computer control enables many cultivation modes. Additionally, we present the construction of the Bacillus subtilis strain ZB designed for bioreactor culture that exhibits a zero-biofilm phenotype. To demonstrate the systems versatility, we performed several experimental cultures with this model organism, including chemostat, turbidostat, medium swap, and a cascade of bioreactors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/669317v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@16b3bbborg.highwire.dtl.DTLVardef@11ea708org.highwire.dtl.DTLVardef@1681769org.highwire.dtl.DTLVardef@d91946_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

The mutational landscape of Bacillus subtilis conditional hypermutators suggests how proofreading inherently skews polymerase error rates

Polymerase errors during DNA replication are a major source of point mutations in genomes. The resulting rate of spontaneous mutation also depends on the counteracting activity of DNA repair mechanisms, with mutator phenotypes appearing constantly and allowing for periods of rapid evolution in nature and in the laboratory. Here, we use the Gram-positive model bacterium Bacillus subtilis to disentangle the contributions of DNA polymerase initial nucleotide selectivity, DNA polymerase proofreading, and mismatch repair (MMR) to the mutation rate. To achieve this, we constructed several conditional hypermutators with a proofreading-deficient allele of polC and/or a deficient allele of mutL and performed mutation accumulation experiments. With their wide range of mutation rates and contrasting mutation profiles, these conditional hypermutators enrich the B. subtilis synthetic biology toolbox for directed evolution. Using mathematical models, we investigated how to interpret the apparent probabilities with which errors escape MMR and proofreading, highlighting the difficulties of working with counts that aggregate potentially heterogeneous mutations and with unknowns about the pathways leading to mutations in the wild-type. Aware of these difficulties, the analysis shows that proofreading prevents partial saturation of the MMR in B. subtilis and that an inherent drawback of proofreading is to skew the net polymerase error rates by amplifying intrinsic biases in nucleotide selectivity.

genomics↗