Hornby, D. P. | Search bioRxiv

bioRxiv2022

Cell Chromatography: Biocompatible chromatographic separation and interrogation of microbial cells

The isolation of pure, single colonies lies at the heart of experimental microbiology. However, a microbial colony typically contains around one million cells at all stages of the life cycle. Here we describe a novel, cell chromatography method that facilitates the capture, purification and interrogation of microbial cells from both single and mixed cultures. The method described relies on, but is not limited to, differences in surface charge to separate bacterial strains. The method is fully biocompatible, leading to no significant loss of cell viability. The chromatographic capture of cells, combined with selective elution methods facilitates a greater level of experimental control over the sample inputs required for downstream high throughput and high sensitivity, analytical methods. The application of the method for interrogating the antibiotic resistance of bacterial strains and for the separation of bacteria from environmental samples is illustrated.

microbiology↗

bioRxiv2020

Systemic mutational rescue in Escherichia coli elicited by a valency dependent, high affinity protein DNA interaction

The controlled formation of sequence specific DNA protein complexes is a fundamental feature of most genetic transactions. Studies of the impact of point mutations on the function of individual components, such as repressors, remains a key aspect of many Systems and Synthetic Biology research programmes. One of the most dramatic systemic consequences of a point mutation is exhibited by the monomeric DNA methyltransferases M.HhaI and M.EcoRII, where substitution of a single, catalytic cysteine by either glycine or alanine, creates a lethal gain of function phenotype. In vivo expression of these point mutants promotes the deposition of high affinity nucleoprotein complexes that arrest replication in vivo, causing cell death. Interestingly, it appears that a systemic response to expression of these mutant enzymes is dramatically enhanced when they are expressed as synthetic dimers. A previously unreported form of "mutational rescue" appears to be triggered as a result of networked crosslinking of host cell DNA resulting from an increased valency of nucleoprotein complex formation. This finding may have significance for developing molecular interventions for the controlled regulation of genome function and for the development of novel antimicrobial and anticancer strategies.

genomics↗